Methods and apparatus for measuring analytes

ABSTRACT

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Applications 61/196,953, 61/198,222, 61/205,626, filed Oct.22, 2008, Nov. 4, 2008 and Jan. 22, 2009, respectively, the entirecontents of all of which are incorporated by reference.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to inventive methods andapparatus relating to detection and measurement of one or more analytesincluding analytes associated with or resulting from a nucleic acidsynthesis reaction.

BACKGROUND

Electronic devices and components have found numerous applications inchemistry and biology (more generally, “life sciences”), especially fordetection and measurement of various chemical and biological reactionsand identification, detection and measurement of various compounds. Onesuch electronic device is referred to as an ion-sensitive field effecttransistor, often denoted in the relevant literature as ISFET (orpHFET). ISFETs conventionally have been explored, primarily in theacademic and research community, to facilitate measurement of thehydrogen ion concentration of a solution (commonly denoted as “pH”).

More specifically, an ISFET is an impedance transformation device thatoperates in a manner similar to that of a MOSFET (Metal OxideSemiconductor Field Effect Transistor), and is particularly configuredto selectively measure ion activity in a solution (e.g., hydrogen ionsin the solution are the “analytes”). A detailed theory of operation ofan ISFET is given in “Thirty years of ISFETOLOGY: what happened in thepast 30 years and what may happen in the next 30 years,” P. Bergveld,Sens. Actuators, 88 (2003), pp. 1-20, which publication is herebyincorporated herein by reference (hereinafter referred to as“Bergveld”).

FIG. 1 illustrates a cross-section of a p-type (p-channel) ISFET 50fabricated using a conventional CMOS (Complementary Metal OxideSemiconductor) process. However, biCMOS (i.e., bipolar and CMOS)processing may also be used, such as a process that would include a PMOSFET array with bipolar structures on the periphery. Alternatively, othertechnologies may be employed wherein a sensing element can be made witha three-terminal devices in which a sensed ion leads to the developmentof a signal that controls one of the three terminals; such technologiesmay also include, for example, GaAs and carbon nanotube technologies.Taking the CMOS example, P-type ISFET fabrication is based on a p-typesilicon substrate 52, in which an n-type well 54 forming a transistor“body” is formed. Highly doped p-type (p+) regions S and D, constitutinga source 56 and a drain 58 of the ISFET, are formed within the n-typewell 54. A highly doped n-type (n+) region B is also formed within then-type well to provide a conductive body (or “bulk”) connection 62 tothe n-type well. An oxide layer 65 is disposed above the source, drainand body connection regions, through which openings are made to provideelectrical connections (via electrical conductors) to these regions; forexample, metal contact 66 serves as a conductor to provide an electricalconnection to the drain 58, and metal contact 68 serves as a conductorto provide a common connection to the source 56 and n-type well 54, viathe highly conductive body connection 62. A polysilicon gate 64 isformed above the oxide layer at a location above a region 60 of then-type well 54, between the source 56 and the drain 58. Because it isdisposed between the polysilicon gate 64 and the transistor body (i.e.,the n-type well), the oxide layer 65 often is referred to as the “gateoxide.”

Like a MOSFET, the operation of an ISFET is based on the modulation ofcharge concentration (and thus channel conductance) caused by a MOS(Metal-Oxide-Semiconductor) capacitance constituted by the polysilicongate 64, the gate oxide 65 and the region 60 of the n-type well 54between the source and the drain. When a negative voltage is appliedacross the gate and source regions (V_(GS)<0 Volts), a “p-channel” 63 iscreated at the interface of the region 60 and the gate oxide 65 bydepleting this area of electrons. This p-channel 63 extends between thesource and the drain, and electric current is conducted through thep-channel when the gate-source potential V_(GS) is negative enough toattract holes from the source into the channel. The gate-sourcepotential at which the channel 63 begins to conduct current is referredto as the transistor's threshold voltage V_(TH) (the transistor conductswhen V_(GS) has an absolute value greater than the threshold voltageV_(TH)). The source is so named because it is the source of the chargecarriers (holes for a p-channel) that flow through the channel 63;similarly, the drain is where the charge carriers leave the channel 63.

In the ISFET 50 of FIG. 1, the n-type well 54 (transistor body), via thebody connection 62, is forced to be biased at a same potential as thesource 56 (i.e., V_(SB)=0 Volts), as seen by the metal contact 68connected to both the source 56 and the body connection 62. Thisconnection prevents forward biasing of the p+ source region and then-type well, and thereby facilitates confinement of charge carriers tothe area of the region 60 in which the channel 63 may be formed. Anypotential difference between the source 56 and the body/n-type well 54(a non-zero source-to-body voltage V_(SB)) affects the threshold voltageV_(TH) of the ISFET according to a nonlinear relationship, and iscommonly referred to as the “body effect,” which in many applications isundesirable.

As also shown in FIG. 1, the polysilicon gate 64 of the ISFET 50 iscoupled to multiple metal layers disposed within one or more additionaloxide layers 75 disposed above the gate oxide 65 to form a “floatinggate” structure 70. The floating gate structure is so named because itis electrically isolated from other conductors associated with theISFET; namely, it is sandwiched between the gate oxide 65 and apassivation layer 72. In the ISFET 50, the passivation layer 72constitutes an ion-sensitive membrane that gives rise to theion-sensitivity of the device. The presence of analytes such as ions inan “analyte solution” 74 (i.e., a solution containing analytes(including ions) of interest or being tested for the presence ofanalytes of interest) in contact with the passivation layer 72,particularly in a sensitive area 78 above the floating gate structure70, alters the electrical characteristics of the ISFET so as to modulatea current flowing through the p-channel 63 between the source 56 and thedrain 58. The passivation layer 72 may comprise any one of a variety ofdifferent materials to facilitate sensitivity to particular ions; forexample, passivation layers comprising silicon nitride or siliconoxynitride, as well as metal oxides such as silicon, aluminum ortantalum oxides, generally provide sensitivity to hydrogen ionconcentration (pH) in the analyte solution 74, whereas passivationlayers comprising polyvinyl chloride containing valinomycin providesensitivity to potassium ion concentration in the analyte solution 74.Materials suitable for passivation layers and sensitive to other ionssuch as sodium, silver, iron, bromine, iodine, calcium, and nitrate, forexample, are known.

With respect to ion sensitivity, an electric potential difference,commonly referred to as a “surface potential,” arises at thesolid/liquid interface of the passivation layer 72 and the analytesolution 74 as a function of the ion concentration in the sensitive area78 due to a chemical reaction (e.g., usually involving the dissociationof oxide surface groups by the ions in the analyte solution 74 inproximity to the sensitive area 78). This surface potential in turnaffects the threshold voltage V_(TH) of the ISFET; thus, it is thethreshold voltage V_(TH) of the ISFET that varies with changes in ionconcentration in the analyte solution 74 in proximity to the sensitivearea 78.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET 50 shown in FIG. 1. With reference again to FIG. 1, a referenceelectrode 76 (a conventional Ag/AgCl electrode) in the analyte solution74 determines the electric potential of the bulk of the analyte solution74 itself and is analogous to the gate terminal of a conventionalMOSFET, as shown in FIG. 2. In a linear or non-saturated operatingregion of the ISFET, the drain current I_(D) is given as:

$\begin{matrix}{{I_{D} = {{\beta\left( {V_{GS} - V_{TH} - {\frac{1}{2}V_{DS}}} \right)}V_{DS}}},} & (1)\end{matrix}$

where V_(DS) is the voltage between the drain and the source, and β is atransconductance parameter (in units of Amps/Volts²) given by:

$\begin{matrix}{{\beta = {\mu \; {C_{ox}\left( \frac{W}{L} \right)}}},} & (2)\end{matrix}$

where μ represents the carrier mobility, C_(ox) is the gate oxidecapacitance per unit area, and the ratio W/L is the width to lengthratio of the channel 63. If the reference electrode 76 provides anelectrical reference or ground (V_(G)=0 Volts), and the drain currentI_(D) and the drain-to-source voltage V_(DS) are kept constant,variations of the source voltage V_(S) of the ISFET directly trackvariations of the threshold voltage V_(TH), according to Eq. (1); thismay be observed by rearranging Eq. (1) as:

$\begin{matrix}{V_{S} = {{- V_{TH}} - {\left( {\frac{I_{D}}{\beta \; V_{DS}} + \frac{V_{DS}}{2}} \right).}}} & (3)\end{matrix}$

Since the threshold voltage V_(TH) of the ISFET is sensitive to ionconcentration as discussed above, according to Eq. (3) the sourcevoltage V_(S) provides a signal that is directly related to the ionconcentration in the analyte solution 74 in proximity to the sensitivearea 78 of the ISFET. More specifically, the threshold voltage V_(TH) isgiven by:

$\begin{matrix}{{V_{TH} = {V_{FB} - \frac{Q_{B}}{C_{ox}} + {2\varphi_{F}}}},} & (4)\end{matrix}$

where VF is the flatband voltage, Q_(B) is the depletion charge in thesilicon and φ_(F) is the Fermi-potential. The flatband voltage in turnis related to material properties such as workfunctions and chargeaccumulation. In the case of an ISFET, with reference to FIGS. 1 and 2,the flatband voltage contains terms that reflect interfaces between 1)the reference electrode 76 (acting as the transistor gate G) and theanalyte solution 74; and 2) the analyte solution 74 and the passivationlayer 72 in the sensitive area 78 (which in turn mimics the interfacebetween the polysilicon gate 64 of the floating gate structure 70 andthe gate oxide 65). The flatband voltage V_(FB) is thus given by:

$\begin{matrix}{{V_{FB} = {E_{ref} - \Psi_{0} + \chi_{sol} - \frac{\Phi_{Si}}{q} - \frac{Q_{ss} + Q_{ox}}{C_{ox}}}},} & (5)\end{matrix}$

where E_(ref) is the reference electrode potential relative to vacuum,Ψ₀ is the surface potential that results from chemical reactions at theanalyte solution/passivation layer interface (e.g., dissociation ofsurface groups in the passivation layer), and χ_(sol) is the surfacedipole potential of the analyte solution 74. The fourth term in Eq. (5)relates to the silicon workfunction (q is the electron charge), and thelast term relates to charge densities at the silicon surface and in thegate oxide. The only term in Eq. (5) sensitive to ion concentration inthe analyte solution 74 is Ψ₀, as the ion concentration in the analytesolution 74 controls the chemical reactions (dissociation of surfacegroups) at the analyte solution/passivation layer interface. Thus,substituting Eq. (5) into Eq. (4), it may be readily observed that it isthe surface potential Ψ₀ that renders the threshold voltage V_(TH)sensitive to ion concentration in the analyte solution 74.

Regarding the chemical reactions at the analyte solution/passivationlayer interface, the surface of a given material employed for thepassivation layer 72 may include chemical groups that may donate protonsto or accept protons from the analyte solution 74, leaving at any giventime negatively charged, positively charged, and neutral sites on thesurface of the passivation layer 72 at the interface with the analytesolution 74. A model for this proton donation/acceptance process at theanalyte solution/passivation layer interface is referred to in therelevant literature as the “Site-Dissociation Model” or the“Site-Binding Model,” and the concepts underlying such a process may beapplied generally to characterize surface activity of passivation layerscomprising various materials (e.g., metal oxides, metal nitrides, metaloxynitrides).

Using the example of a metal oxide for purposes of illustration, thesurface of any metal oxide contains hydroxyl groups that may donate aproton to or accept a proton from the analyte to leave negatively orpositively charged sites, respectively, on the surface. The equilibriumreactions at these sites may be described by:

AOH⇄AO ⁻ +H _(s) ⁺  (6)

AOH2⁺ ⇄AOH+H _(s) ⁺  (7)

where A denotes an exemplary metal, H_(s) ⁺ represents a proton in theanalyte solution 74. Eq. (6) describes proton donation by a surfacegroup, and Eq. (7) describes proton acceptance by a surface group. Itshould be appreciated that the reactions given in Eqs. (6) and (7) alsoare present and need to be considered in the analysis of a passivationlayer comprising metal nitrides, together with the equilibrium reaction:

ANH ₃ ⁺ ⇄ANH ₂ +H ⁺  (7b)

wherein Eq. (7b) describes another proton acceptance equilibriumreaction. For purposes of the present discussion however, again only theproton donation and acceptance reactions given in Eqs. (6) and (7) areinitially considered to illustrate the relevant concepts.

Based on the respective forward and backward reaction rate constants foreach equilibrium reaction, intrinsic dissociation constants K_(a) (forthe reaction of Eq. (6)) and K_(b) (for the reaction of Eq. (7)) may becalculated that describe the equilibrium reactions. These intrinsicdissociation constants in turn may be used to determine a surface chargedensity σ₀ (in units of Coulombs/unit area) of the passivation layer 72according to:

σ₀ =−qB  (8)

where the term B denotes the number of negatively charged surface groupsminus the number of positively charged surface groups per unit area,which in turn depends on the total number of proton donor/acceptor sitesper unit area N_(S) on the passivation layer surface, multiplied by afactor relating to the intrinsic dissociation constants K_(a) and K_(b)of the respective proton donation and acceptance equilibrium reactionsand the surface proton activity (or pHs). The effect of a small changein surface proton activity (pHs) on the surface charge density is givenby:

$\begin{matrix}{{\frac{\partial\sigma_{0}}{{\partial p}\; H_{s}} = {{{- q}\frac{\partial B}{{\partial p}\; H_{s}}} = {{- q}\; \beta_{int}}}},} & (9)\end{matrix}$

where β_(int) is referred to as the “intrinsic buffering capacity” ofthe surface. It should be appreciated that since the values of N_(S),K_(a) and K_(b) are material dependent, the intrinsic buffering capacityβ_(int) of the surface similarly is material dependent.

The fact that ionic species in the analyte solution 74 have a finitesize and cannot approach the passivation layer surface any closer thanthe ionic radius results in a phenomenon referred to as a “double layercapacitance” proximate to the analyte solution/passivation layerinterface. In the Gouy-Chapman-Stern model for the double layercapacitance as described in Bergveld, the surface charge density σ₀ isbalanced by an equal but opposite charge density in the analyte solution74 at some position from the surface of the passivation layer 72. Thesetwo parallel opposite charges form a so-called “double layercapacitance” C_(dl) (per unit area), and the potential difference acrossthe capacitance C_(dl) is defined as the surface potential Ψ₀, accordingto:

σ₀ =C _(dl)Ψ₀=−σ_(dl)  (10)

where σ_(dl) is the charge density on the analyte solution side of thedouble layer capacitance. This charge density σ_(dl) in turn is afunction of the concentration of all ion species or other analytespecies (i.e., not just protons) in the bulk analyte solution 74; inparticular, the surface charge density can be balanced not only byhydrogen ions but other ion species (e.g., Na⁺, K⁺) in the bulk analytesolution.

In the regime of relatively lower ionic strengths (e.g., <1 mole/liter),the Debye theory may be used to describe the double layer capacitanceC_(dl) according to:

$\begin{matrix}{C_{dl} = \frac{k\; ɛ_{0}}{\lambda}} & (11)\end{matrix}$

where k is the dielectric constant ∈/∈₀ (for relatively lower ionicstrengths, the dielectric constant of water may be used), and λ is theDebye screening length (i.e., the distance over which significant chargeseparation can occur). The Debye length λ is in turn inverselyproportional to the square root of the strength of the ionic species inthe analyte solution, and in water at room temperature is given by:

$\begin{matrix}{\lambda = {\frac{0.3\mspace{14mu} {nm}}{\sqrt{I}}.}} & (12)\end{matrix}$

The ionic strength I of the bulk analyte is a function of theconcentration of all ionic species present, and is given by:

$\begin{matrix}{{I = {\frac{1}{2}{\sum\limits_{s}{z_{s}^{2}c_{s}}}}},} & (13)\end{matrix}$

where z_(s) is the charge number of ionic species s and c_(s) is themolar concentration of ionic species s. Accordingly, from Eqs. (10)through (13), it may be observed that the surface potential is largerfor larger Debye screening lengths (i.e., smaller ionic strengths).

The relation between pH values present at the analytesolution/passivation layer interface and in the bulk solution isexpressed in the relevant literature by Boltzman statistics with thesurface potential Ψ₀ as a parameter:

$\begin{matrix}{\left( {{p\; H_{s}} - {p\; H_{B}}} \right) = {\frac{q\; \Psi_{0}}{kT}.}} & (14)\end{matrix}$

From Eqs. (9), (10) and (14), the sensitivity of the surface potentialΨ₀ particularly to changes in the bulk pH of the analyte solution (i.e.,“pH sensitivity”) is given by:

$\begin{matrix}{{\frac{\Delta \; \Psi_{0}}{\Delta \; {pH}} = {{- 2.3}\frac{k\; T}{q}\alpha}},} & (15)\end{matrix}$

where the parameter α is a dimensionless sensitivity factor that variesbetween zero and one and depends on the double layer capacitance C_(dl)and the intrinsic buffering capacity of the surface β_(int) as discussedabove in connection with Eq. (9). In general, passivation layermaterials with a high intrinsic buffering capacity β_(int) render thesurface potential Ψ₀ less sensitive to concentration in the analytesolution 74 of ionic species other than protons (e.g., a is maximized bya large β_(int)). From Eq. (15), at a temperature T of 298 degreesKelvin, it may be appreciated that a theoretical maximum pH sensitivityof 59.2 mV/pH may be achieved at α=1. From Eqs. (4) and (5), as notedabove, changes in the ISFET threshold voltage V_(TH) directly trackchanges in the surface potential o; accordingly, the pH sensitivity ofan ISFET given by Eq. (15) also may be denoted and referred to herein asΔV_(TH) for convenience. In exemplary conventional ISFETs employing asilicon nitride or silicon oxynitride passivation layer 72 forpH-sensitivity, pH sensitivities ΔV_(TH) (i.e., a change in thresholdvoltage with change in pH of the analyte solution 74) over a range ofapproximately 30 mV/pH to 60 mV/pH have been observed experimentally.

Another noteworthy metric in connection with ISFET pH sensitivityrelates to the bulk pH of the analyte solution 74 at which there is nonet surface charge density σ₀ and, accordingly, a surface potential Ψ₀of zero volts. This pH is referred to as the “point of zero charge” anddenoted as pH_(pzc). With reference again to Eqs. (8) and (9), like theintrinsic buffering capacity β_(int), pH_(pzc) is a material dependentparameter. From the foregoing, it may be appreciated that the surfacepotential at any given bulk pH_(B) of the analyte solution 74 may becalculated according to:

$\begin{matrix}{{\Psi_{0}\left( {p\; H_{B}} \right)} = {\left( {{p\; H_{B}} - {p\; H_{pzc}}} \right){\frac{\Delta \; \Psi_{0}}{\Delta \; {pH}}.}}} & (16)\end{matrix}$

Table 1 below lists various metal oxides and metal nitrides and theircorresponding points of zero charge (pH_(pzc)), pH sensitivities(ΔV_(TH)), and theoretical maximum surface potential at a pH of 9:

TABLE 1 Oxide/ Theoretical Ψ₀ Metal Nitride pH_(pzc) ΔV_(TH) (mV/pH)(mV) @ pH = 9 Al Al₂O₃ 9.2  54.5 (35° C.) −11 Zr ZrO₂ 5.1 50 150 Ti TiO₂5.5 57.4-62.3 201 (32° C., pH 3-11) Ta Ta₂O₅ 2.9, 2.8 62.87 (35° C.) 384Si Si₃N₄ 4.6, 6-7 56.94 (25° C.) 251 Si SiO₂ 2.1 43 297 Mo MoO₃ 1.8-2.148-59 396 Hf HfO₂ 7-4-7.6 50-58 81.2 W WO₂ 0.3, 0.43, 0.5 50 435

Prior research efforts to fabricate ISFETs for pH measurements based onconventional CMOS processing techniques typically have aimed to achievehigh signal linearity over a pH range from 1-14. Using an exemplarythreshold sensitivity of approximately 50 mV/pH, and considering Eq. (3)above, this requires a linear operating range of approximately 700 mVfor the source voltage V_(S). As discussed above in connection with FIG.1, the threshold voltage V_(TH) of ISFETs (as well as MOSFETs) isaffected by any voltage V_(SB) between the source and the body (n-typewell 54). More specifically, the threshold voltage V_(TH) is a nonlinearfunction of a nonzero source-to-body voltage V_(SB). Accordingly, so asto avoid compromising linearity due to a difference between the sourceand body voltage potentials (i.e., to mitigate the “body effect”), asshown in FIG. 1 the source 56 and body connection 62 of the ISFET 50often are coupled to a common potential via the metal contact 68. Thisbody-source coupling also is shown in the electric circuitrepresentation of the ISFET 50 shown in FIG. 2.

While the foregoing discussion relates primarily to a steady stateanalysis of ISFET response based on the equilibrium reactions given inEqs. (6) and (7), the transient or dynamic response of a conventionalISFET to an essentially instantaneous change in ionic strength of theanalyte solution 74 (e.g., a stepwise change in proton or other ionicspecies concentration) has been explored in some research efforts. Oneexemplary treatment of ISFET transient or dynamic response is found in“ISFET responses on a stepwise change in electrolyte concentration atconstant pH,” J. C. van Kerkof, J. C. T. Eijkel and P. Bergveld, Sensorsand Actuators B, 18-19 (1994), pp. 56-59, which is incorporated hereinby reference.

For ISFET transient response, a stepwise change in the concentration ofone or more ionic species in the analyte solution in turn essentiallyinstantaneously changes the charge density σ_(dl) on the analytesolution side of the double layer capacitance C_(dl). Because theinstantaneous change in charge density σ_(dl) is faster than thereaction kinetics at the surface of the passivation layer 72, thesurface charge density σ₀ initially remains constant, and the change inion concentration effectively results in a sudden change in the doublelayer capacitance C_(dl). From Eq. (10), it may be appreciated that sucha sudden change in the capacitance C_(dl) at a constant surface chargedensity σ₀ results in a corresponding sudden change in the surfacepotential Ψ₀. FIG. 2A illustrates this phenomenon, in which anessentially instantaneous or stepwise increase in ion concentration inthe analyte solution, as shown in the top graph, results in acorresponding change in the surface potential o, as shown in the bottomgraph of FIG. 2A. After some time, as the passivation layer surfacegroups react to the stimulus (i.e., as the surface charge densityadjusts), the system returns to some equilibrium point, as illustratedby the decay of the ISFET response “pulse” 79 shown in the bottom graphof FIG. 2A. The foregoing phenomenon is referred to in the relevantliterature (and hereafter in this disclosure) as an “ion-step” response.

As indicated in the bottom graph of FIG. 2A, an amplitude ΔΨ₀ of theion-step response 79 may be characterized by:

$\begin{matrix}{{{\Delta \; \Psi_{0}} = {{\Psi_{1} - \Psi_{2}} = {{\frac{\sigma_{0}}{C_{{dl},1}} - \frac{\sigma_{0}}{C_{{dl},2}}} = {\Psi_{1}\left( {1 - \frac{C_{{dl},1}}{C_{{dl},2}}} \right)}}}},} & (17)\end{matrix}$

where Ψ₁ is an equilibrium surface potential at an initial ionconcentration in the analyte solution, C_(dl.1) is the double layercapacitance per unit area at the initial ion concentration, Ψ₂ is thesurface potential corresponding to the ion-step stimulus, and C_(dl.2)is the double layer capacitance per unit area based on the ion-stepstimulus. The time decay profile 81 associated with the response 79 isdetermined at least in part by the kinetics of the equilibrium reactionsat the analyte solution/passivation layer interface (e.g., as given byEqs. (6) and (7) for metal oxides, and also Eq. (7b) for metalnitrides). One instructive treatment in this regard is provided by“Modeling the short-time response of ISFET sensors,” P. Woias et al.,Sensors and Actuators B, 24-25 (1995) 211-217 (hereinafter referred toas “Woias”), which publication is incorporated herein by reference.

In the Woias publication, an exemplary ISFET having a silicon nitridepassivation layer is considered. A system of coupled non-lineardifferential equations based on the equilibrium reactions given by Eqs.(6), (7), and (7a) is formulated to describe the dynamic response of theISFET to a step (essentially instantaneous) change in pH; morespecifically, these equations describe the change in concentration overtime of the various surface species involved in the equilibriumreactions, based on the forward and backward rate constants for theinvolved proton acceptance and proton donation reactions and how changesin analyte pH affect one or more of the reaction rate constants.Exemplary solutions, some of which include multiple exponentialfunctions and associated time constants, are provided for theconcentration of each of the surface ion species as a function of time.In one example provided by Woias, it is assumed that the proton donationreaction given by Eq. (6) dominates the transient response of thesilicon nitride passivation layer surface for relatively small stepchanges in pH, thereby facilitating a mono-exponential approximation forthe time decay profile 81 of the response 79 according to:

Ψ₀(t)=ΔΨ₀ e ^(−1/τ)  (18)

where the exponential function essentially represents the change insurface charge density as a function of time. In Eq. (16), the timeconstant τ is both a function of the bulk pH and material parameters ofthe passivation layer, according to:

τ=τ₀×10^(pH/2)  (19)

where τ₀ denotes a theoretical minimum response time that only dependson material parameters. For silicon nitride, Woias provides exemplaryvalues for τ₀ on the order of 60 microseconds to 200 microseconds. Forpurposes of providing an illustrative example, using τ₀=60 microsecondsand a bulk pH of 9, the time constant τ given by Eq. (19) is 1.9seconds. Exemplary values for other types of passivation materials maybe found in the relevant literature and/or determined empirically.

Previous efforts to fabricate two-dimensional arrays of ISFETs based onthe ISFET design of FIG. 1 have resulted in a maximum of 256 ISFETsensor elements (or “pixels”) in an array (i.e., a 16 pixel by 16 pixelarray). Exemplary research in ISFET array fabrication is reported in thepublications “A large transistor-based sensor array chip for directextracellular imaging,” M. J. Milgrew, M. O. Riehle, and D. R. S.Cumming, Sensors and Actuators, B: Chemical, 111-112, (2005), pp.347-353, and “The development of scalable sensor arrays using standardCMOS technology,” M. J. Milgrew, P. A. Hammond, and D. R. S. Cumming,Sensors and Actuators, B: Chemical, 103, (2004), pp. 37-42, whichpublications are incorporated herein by reference and collectivelyreferred to hereafter as “Milgrew et al.” Other research effortsrelating to the realization of ISFET arrays are reported in thepublications “A very large integrated pH-ISFET sensor array chipcompatible with standard CMOS processes,” T. C. W. Yeow, M. R. Haskard,D. E. Mulcahy, H. I. Seo and D. H. Kwon, Sensors and Actuators B:Chemical, 44, (1997), pp. 434-440 and “Fabrication of a two-dimensionalpH image sensor using a charge transfer technique,” Hizawa, T., Sawada,K., Takao, H., Ishida, M., Sensors and Actuators, B: Chemical 117 (2),2006, pp. 509-515, which publications also are incorporated herein byreference.

FIG. 3 illustrates one column 85 _(j) of a two-dimensional ISFET arrayaccording to the design of Milgrew et al. The column 85 _(j) includessixteen (16) pixels 80 ₁ through 80 ₁₆ and, as discussed further belowin connection with FIG. 7, a complete two-dimensional array includessixteen (16) such columns 85 _(j) (j=1, 2, 3, . . . 16) arranged side byside. As shown in FIG. 3, a given column 85 _(j) includes a currentsource I_(SOURCEj) that is shared by all pixels of the column, and ISFETbias/readout circuitry 82 _(j) (including current sink I_(SINKj)) thatis also shared by all pixels of the column. Each ISFET pixel 80 ₁through 80 ₁₆ includes a p-channel ISFET 50 having an electricallycoupled source and body (as shown in FIGS. 1 and 2), plus two switchesS1 and S2 that are responsive to one of sixteen row select signals(RSEL₁ through RSEL₁₆, and their complements). As discussed below inconnection with FIG. 7, a row select signal and its complement aregenerated simultaneously to “enable” or select a given pixel of thecolumn 85 _(j), and such signal pairs are generated in some sequence tosuccessively enable different pixels of the column one at a time.

As shown in FIG. 3, the switch S2 of each pixel 80 in the design ofMilgrew et al. is implemented as a conventional n-channel MOSFET thatcouples the current source I_(SOURCEj) to the source of the ISFET 50upon receipt of the corresponding row select signal. The switch S1 ofeach pixel 80 is implemented as a transmission gate, i.e., a CMOS pairincluding an n-channel MOSFET and a p-channel MOSFET, that couples thesource of the ISFET 50 to the bias/readout circuitry 82 _(j) uponreceipt of the corresponding row select signal and its complement. Anexample of the switch S1 ₁ of the pixel 80 ₁ is shown in FIG. 4, inwhich the p-channel MOSFET of the transmission gate is indicated as S1_(1P) and the n-channel MOSFET is indicated as S1 _(1N). In the designof Milgrew et al., a transmission gate is employed for the switch S1 ofeach pixel so that, for an enabled pixel, any ISFET source voltagewithin the power supply range V_(DD) to V_(SS) may be applied to thebias/readout circuitry 82 _(j) and output by the column as the signalV_(Sj). From the foregoing, it should be appreciated that each pixel 80in the ISFET sensor array design of Milgrew et al. includes fourtransistors, i.e., a p-channel ISFET, a CMOS-pair transmission gateincluding an n-channel MOSFET and a p-channel MOSFET for switch S1, andan n-channel MOSFET for switch S2.

As also shown in FIG. 3, the bias/readout circuitry 82 _(j) employs asource-drain follower configuration in the form of a Kelvin bridge tomaintain a constant drain-source voltage V_(DSj) and isolate themeasurement of the source voltage V_(Sj) from the constant drain currentI_(SOURCEj) for the ISFET of an enabled pixel in the column 85 _(j). Tothis end, the bias/readout circuitry 82 _(j) includes two operationalamplifiers A1 and A2, a current sink I_(SINKj) and a resistor R_(SDj).The voltage developed across the resistor R_(SDj) due to the currentI_(SINKj) flowing through the resistor is forced by the operationalamplifiers to appear across the drain and source of the ISFET of anenabled pixel as a constant drain-source voltage V_(DSj). Thus, withreference again to Eq. (3), due to the constant V_(DSj) and the constantI_(SOURCEj), the source voltage V_(Sj) of the ISFET of the enabled pixelprovides a signal corresponding to the ISFETs threshold voltage V_(TH),and hence a measurement of pH in proximity to the ISFETs sensitive area(see FIG. 1). The wide dynamic range for the source voltage V_(Sj)provided by the transmission gate S1 ensures that a full range of pHvalues from 1-14 may be measured, and the source-body connection of eachISFET ensures sufficient linearity of the ISFETs threshold voltage overthe full pH measurement range.

In the column design of Milgrew et al. shown in FIG. 3, it should beappreciated that for the Kelvin bridge configuration of the columnbias/readout circuitry 82 _(j) to function properly, a p-channel ISFET50 as shown in FIG. 1 must be employed in each pixel; more specifically,an alternative implementation based on the Kelvin bridge configurationis not possible using an n-channel ISFET. With reference again to FIG.1, for an n-channel ISFET based on a conventional CMOS process, then-type well 54 would not be required, and highly doped n-type regionsfor the drain and source would be formed directly in the p-type siliconsubstrate 52 (which would constitute the transistor body). For n-channelFET devices, the transistor body typically is coupled to electricalground. Given the requirement that the source and body of an ISFET inthe design of Milgrew et al. are electrically coupled together tomitigate nonlinear performance due to the body effect, this would resultin the source of an n-channel ISFET also being connected to electricalground (i.e., V_(S)=V_(B)=0 Volts), thereby precluding any useful outputsignal from an enabled pixel. Accordingly, the column design of Milgrewet al. shown in FIG. 3 requires p-channel ISFETs for proper operation.

It should also be appreciated that in the column design of Milgrew etal. shown in FIG. 3, the two n-channel MOSFETs required to implement theswitches S1 and S2 in each pixel cannot be formed in the n-type well 54shown in FIG. 1, in which the p-channel ISFET for the pixel is formed;rather, the n-channel MOSFETs are formed directly in the p-type siliconsubstrate 52, beyond the confines of the n-type well 54 for the ISFET.FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of the p-type silicon substrate 52corresponding to one pixel 80 of the column 85 j shown in FIG. 3, inwhich the n-type well 54 containing the drain 58, source 56 and bodyconnection 62 of the ISFET 50 is shown alongside a first n-channelMOSFET corresponding to the switch S2 and a second n-channel MOSFET S1_(1N) constituting one of the two transistors of the transmission gateS1 ₁ shown in FIG. 4.

Furthermore, in the design of Milgrew et al., the p-channel MOSFETrequired to implement the transmission gate S1 in each pixel (e.g., seeS1 _(1P) in FIG. 4) cannot be formed in the same n-type well in whichthe p-channel ISFET 50 for the pixel is formed. In particular, becausethe body and source of the p-channel ISFET are electrically coupledtogether, implementing the p-channel MOSFET S1 _(1P) in the same n-wellas the p-channel ISFET 50 would lead to unpredictable operation of thetransmission gate, or preclude operation entirely. Accordingly, twoseparate n-type wells are required to implement each pixel in the designof Milgrew et al. FIG. 6 is a diagram similar to FIG. 5, showing across-section of another portion of the p-type silicon substrate 52corresponding to one pixel 80, in which the n-type well 54 correspondingto the ISFET 50 is shown alongside a second n-type well 55 in which isformed the p-channel MOSFET S1 _(1P) constituting one of the twotransistors of the transmission gate S1 ₁ shown in FIG. 4. It should beappreciated that the drawings in FIGS. 5 and 6 are not to scale and maynot exactly represent the actual layout of a particular pixel in thedesign of Milgrew et al.; rather these figures are conceptual in natureand are provided primarily to illustrate the requirements of multiplen-wells, and separate n-channel MOSFETs fabricated outside of then-wells, in the design of Milgrew et al.

The array design of Milgrew et al. was implemented using a 0.35micrometer (μm) conventional CMOS fabrication process. In this process,various design rules dictate minimum separation distances betweenfeatures. For example, according to the 0.35 μm CMOS design rules, withreference to FIG. 6, a distance “a” between neighboring n-wells must beat least three (3) micrometers. A distance “a/2” also is indicated inFIG. 6 to the left of the n-well 54 and to the right of the n-well 55 toindicate the minimum distance required to separate the pixel 80 shown inFIG. 6 from neighboring pixels in other columns to the left and right,respectively. Additionally, according to typical 0.35 μm CMOS designrules, a distance “b” shown in FIG. 6 representing the width incross-section of the n-type well 54 and a distance “c” representing thewidth in cross-section of the n-type well 55 are each on the order ofapproximately 3 μm to 4 μm (within the n-type well, an allowance of 1.2μm is made between the edge of the n-well and each of the source anddrain, and the source and drain themselves have a width on the order of0.7 μm). Accordingly, a total distance “d” shown in FIG. 6 representingthe width of the pixel 80 in cross-section is on the order ofapproximately 12 μm to 14 μm. In one implementation, Milgrew et al.report an array based on the column/pixel design shown in FIG. 3comprising geometrically square pixels each having a dimension of 12.8μm by 12.8 μm.

In sum, the ISFET pixel design of Milgrew et al. is aimed at ensuringaccurate hydrogen ion concentration measurements over a pH range of1-14. To ensure measurement linearity, the source and body of eachpixel's ISFET are electrically coupled together. To ensure a full rangeof pH measurements, a transmission gate S is employed in each pixel totransmit the source voltage of an enabled pixel. Thus, each pixel ofMilgrew's array requires four transistors (p-channel ISFET, p-channelMOSFET, and two n-channel MOSFETs) and two separate n-wells (FIG. 6).Based on a 0.35 micrometer conventional CMOS fabrication process andcorresponding design rules, the pixels of such an array have a minimumsize appreciably greater than 10 μm, i.e., on the order of approximately12 μm to 14 μm.

FIG. 7 illustrates a complete two-dimensional pixel array 95 accordingto the design of Milgrew et al., together with accompanying row andcolumn decoder circuitry and measurement readout circuitry. The array 95includes sixteen columns 85 ₁ through 85 ₁₆ of pixels, each columnhaving sixteen pixels as discussed above in connection with FIG. 3(i.e., a 16 pixel by 16 pixel array). A row decoder 92 provides sixteenpairs of complementary row select signals, wherein each pair of rowselect signals simultaneously enables one pixel in each column 85 ₁through 85 ₁₆ to provide a set of column output signals from the array95 based on the respective source voltages V_(S1) through V_(S16) of theenabled row of ISFETs. The row decoder 92 is implemented as aconventional four-to-sixteen decoder (i.e., a four-bit binary inputROW₁-ROW₄ to select one of 2⁴ outputs). The set of column output signalsV_(S1) through V_(S16) for an enabled row of the array is applied toswitching logic 96, which includes sixteen transmission gates S1 throughS16 (one transmission gate for each output signal). As above, eachtransmission gate of the switching logic 96 is implemented using ap-channel MOSFET and an n-channel MOSFET to ensure a sufficient dynamicrange for each of the output signals V_(S1) through V_(S16). The columndecoder 94, like the row decoder 92, is implemented as a conventionalfour-to-sixteen decoder and is controlled via the four-bit binary inputCOL₁-COL₄ to enable one of the transmission gates S1 through S16 of theswitching logic 96 at any given time, so as to provide a single outputsignal V_(S) from the switching logic 96. This output signal V_(S) isapplied to a 10-bit analog to digital converter (ADC) 98 to provide adigital representation D₁-D₁₀ of the output signal V_(S) correspondingto a given pixel of the array.

As noted earlier, individual ISFETs and arrays of ISFETs similar tothose discussed above have been employed as sensing devices in a varietyof chemical and biological applications. In particular, ISFETs have beenemployed as pH sensors in the monitoring of various processes involvingnucleic acids such as DNA. Some examples of employing ISFETs in variouslife-science related applications are given in the followingpublications, each of which is incorporated herein by reference: MassimoBarbaro, Annalisa Bonfiglio, Luigi Raffo, Andrea Alessandrini, PaoloFacci and Imrich Barák, “Fully electronic DNA hybridization detection bya standard CMOS biochip,” Sensors and Actuators B: Chemical, Volume 118,Issues 1-2, 2006, pp. 41-46; Toshinari Sakurai and Yuzuru Husimi,“Real-time monitoring of DNA polymerase reactions by a micro ISFET pHsensor,” Anal. Chem., 64(17), 1992, pp 1996-1997; S. Purushothaman, C.Toumazou, J. Georgiou, “Towards fast solid state DNA sequencing,”Circuits and Systems, vol. 4, 2002, pp. IV-169 to IV-172; S.Purushothaman, C. Toumazou, C. P. Ou, “Protons and single nucleotidepolymorphism detection: A simple use for the Ion Sensitive Field EffectTransistor,” Sensors and Actuators B: Chemical, Vol. 114, no. 2, 2006,pp. 964-968; A. L. Simonian, A. W. Flounders, J. R. Wild, “FET-BasedBiosensors for The Direct Detection of Organophosphate Neurotoxins,”Electroanalysis, Vol. 16, No. 22, 2004, pp. 1896-1906; C. Toumazou, S.Purushothaman, “Sensing Apparatus and Method,” United States PatentApplication 2004-0134798, published Jul. 15, 2004; and T. W. Koo, S.Chan, X. Su, Z. Jingwu, M. Yamakawa, V. M. Dubin, “Sensor Arrays andNucleic Acid Sequencing Applications,” United States Patent Application2006-0199193, published Sep. 7, 2006.

In general, the development of rapid and sensitive nucleic acidsequencing methods utilizing automated DNA sequencers has significantlyadvanced the understanding of biology. The term “sequencing” refers tothe determination of a primary structure (or primary sequence) of anunbranched biopolymer, which results in a symbolic linear depictionknown as a “sequence” that succinctly summarizes much of theatomic-level structure of the sequenced molecule. Nucleic acid (such asDNA) sequencing particularly refers to the process of determining thenucleotide order of a given nucleic acid fragment. Analysis of entiregenomes of viruses, bacteria, fungi, animals and plants is now possible,but such analysis generally is limited due to the cost and time requiredto sequence such large genomes. Moreover, present conventionalsequencing methods are limited in terms of their accuracy, the length ofindividual templates that can be sequenced, and the rate of sequencedetermination.

Despite improvements in sample preparation and sequencing technologies,none of the present conventional sequencing strategies, including thoseto date that may involve ISFETs, has provided the cost reductionsrequired to increase throughput to levels required for analysis of largenumbers of individual human genomes. The ability to sequence many humangenomes facilitates an analysis of the genetic basis underlying disease(e.g., such as cancer) and aging, for example. Some recent efforts havemade significant gains in both the ability to prepare genomes forsequencing and to sequence large numbers of templates simultaneously.However, these and other efforts are still limited by the relativelylarge size of the reaction volumes, as well as the need for specialnucleotide analogues, and complex enzymatic or fluorescent methods to“read out” nucleotide sequence.

SUMMARY

Aspects of the invention relate in part to the use of large arrays ofchemically sensitive FETs (chemFETs) or more specifically ISFETs formonitoring reactions, including for example nucleic acid (e.g., DNA)sequencing reactions, based on monitoring analytes present, generated orused during a reaction. More generally, arrays including large arrays ofchemFETs may be employed to detect and measure static and/or dynamicamounts or concentrations of a variety of analytes (e.g., hydrogen ions,other ions, non-ionic molecules or compounds, etc.) in a variety ofchemical and/or biological processes (e.g., biological or chemicalreactions, cell or tissue cultures or monitoring, neural activity,nucleic acid sequencing, etc.) in which valuable information may beobtained based on such analyte measurements. Such chemFET arrays may beemployed in methods that detect analytes and/or methods that monitorbiological or chemical processes via changes in charge at the chemFETsurface. Accordingly, the systems and methods shown herein provide usesfor chemFET arrays that involve detection of analytes in solution and/ordetection of change in charge bound to the chemFET surface.

Methods are presented for maintaining or increasing signal (and thussignal-to-noise ratio) when using very large chemFET arrays, and inparticular when increasing the density of a chemFET array (andconcomitantly decreasing the area of any single chemFET within thearray). It has been found that as chemFET area decreases in order toaccommodate an ever increasing number of sensors on a given array, thesignal that can be obtained from a single chemFET may in some instancesdecrease. The invention provides in some aspects and embodiments methodsfor overcoming this limitation.

Of particular importance is the ability to increase signal during anucleic acid synthesis reaction, and more particularly increasing signalattributable to hydrogen ions that are generated during such a reaction.

In this context, some methods of the invention involve increasing theefficiency with which released (or generated) hydrogen ions aredetected. It has been determined in the course of our work that releasedhydrogen ions may be sequestered in a reaction chamber that overlays thechemFET, thereby precluding their detection by the chemFET. Thisdisclosure therefore provides in some aspects methods and compositionsfor reducing buffering capacity of the solution within which suchreactions are carried out or reducing buffering capacity of solidsupports that are in contact with such solution. In this way, a greaterproportion of the hydrogen ions released during a nucleic acid synthesisreaction (such as one that is part of a sequencing-by-synthesis process)are detected by the chemFET rather than being for example sequestered bybuffering components in the reaction solution or chamber.

Additionally or alternatively, aspects of the invention that monitorand/or measure hydrogen ion release (or pH) may be performed in anenvironment with reduced (i.e., no, low or limited) buffering capacityso as to maximally detect released hydrogen ions. As an example, theinvention provides a method for synthesizing a nucleic acid comprisingincorporating nucleotides into a nucleic acid in an environment with noor limited buffering capacity. Examples of an environment with reducedbuffering capacity (or activity) include one that lacks a buffer, onethat includes a buffer (or buffering) inhibitor, and one in which pHchanges on the order of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9. or 1.0 pH units are detectable for example via a chemFET andmore particularly an ISFET. The method may be performed in a solution ora reaction chamber that is in contact with or capacitively coupled to achemFET such as an ISFET. The chemFET (or ISFET) and/or reaction chambermay be in array of chemFETs or reaction chambers, respectively. Thereactions are typically carried out at a pH (or a pH range) at which thepolymerase is active. An exemplary pH range is 6-9.5, although theinvention is not so limited.

In a related aspect, there is shown a method for sequencing a nucleicacid comprising contacting and incorporating known nucleotides into aplurality of identical nucleic acids in a reaction chamber in contactwith or capacitively coupled to an ISFET, wherein the nucleic acids arecovalently bound to a single bead in the reaction chamber, and detectinghydrogen ions released upon nucleotide incorporation in the presence ofno or limited buffering activity. In other embodiments, the single beadis at least 50%, at least 60%, at least 70%, or at least 80% saturatedwith nucleic acids. In some embodiments, the single bead is at least 90%saturated with nucleic acids. In still other embodiments, the singlebead is at least 95% saturated with nucleic acids. The bead may have adiameter of about 1 micron to about 10 microns, or about 1 micron toabout 7 microns, or about 1 micron to about 5 microns, including adiameter of about 1 micron, about 2 microns, about 3 microns, about 4microns, about 5 microns, about 6 microns, about 7 microns, about 8microns, about 9 microns, or about 10 microns.

In these and in other aspects and embodiments, the chemFET or ISFETarrays may comprise 256 chemFETs or ISFETs. The chemFET or ISFET arraymay have a center-to-center spacing (between adjacent chemFETs orISFETs) of 1-10 microns. In some embodiments, the center-to-centerspacing is about 9 microns, about 8 microns, about 7 microns, about 6microns, about 5 microns, about 4 microns, about 3 microns, about 2microns or about 1 micron. In particular embodiments, thecenter-to-center spacing is about 5.1 microns or about 2.8 microns. Invarious embodiments, the chemFET or ISFET comprises a passivation layerthat is or is not bound to a nucleic acid.

In these and in other aspects and embodiments, the reaction chamber maycomprise a solution having no buffer or low buffer concentration. Themethods described herein may be performed in a weak buffer.Alternatively or additionally, the reaction chamber may comprise asolution having a buffering inhibitor. The reaction chamber may or maynot comprise packing beads. In some embodiments, the reaction chamber isin contact with a single ISFET. In some embodiments, the reactionchamber has a volume of equal to or less than about 1 picoliter (pL).

In some embodiments, the nucleic acids are sequencing primers. Thenucleic acids may be hybridized to template nucleic acids or toconcatemers of identical template nucleic acids. In still otherembodiments the nucleic acids are self-priming template nucleic acids.In still other embodiments, the nucleic acids are nicked double-strandednucleic acids.

In these and in other aspects and embodiments, the nucleotides may beunblocked. In some embodiments, the nucleotides are not extrinsicallylabeled. In some embodiments, nucleic acids are synthesized ornucleotides are incorporated using a polymerase that is free insolution. In some embodiments, nucleic acids are synthesized ornucleotides are incorporated using a polymerase that is immobilized. Inrelated embodiments, the polymerase is immobilized to the bead, or to aseparate bead. The polymerase may be provided in a mixture ofpolymerases, including a mixture of 2, 3 or more polymerases.

In another aspect, there is provided a method for synthesizing a nucleicacid comprising incorporating nucleotides into a nucleic acid in thepresence of a buffering inhibitor. In one embodiment, the method furthercomprises detecting incorporation of nucleotides by detecting hydrogenion release.

In another aspect, there is provided a method for determiningincorporation of a nucleotide triphosphate into a newly synthesizednucleic acid comprising combining a known nucleotide triphosphate, atemplate/primer hybrid, a buffering inhibitor and a polymerase, in asolution in contact with or capacitively coupled to a chemFET, anddetecting a signal at the chemFET, wherein detection of the signalindicates incorporation of the known nucleotide triphosphate into thenewly synthesized nucleic acid. In one embodiment, the signal indicatesrelease of hydrogen ions as a result of nucleotide incorporation. Invarious embodiments, the nucleic acid is a plurality of identicalnucleic acids, the nucleotide triphosphates are a plurality ofnucleotide triphosphates, and the hybrids are a plurality of hybrids.

The buffering inhibitor may be a plurality of random sequenceoligoribonucleotides such as but not limited to RNA hexamers, or it maybe a sulfonic acid surfactant such as but not limited to poly(ethyleneglycol) 4-nonylphenyl 3-sulfopropyl ether (PNSE) or a salt thereof, orit may be poly(styrenesulfonic acid), poly(diallydimethylammonium), ortetramethyl ammonium, or a salt thereof.

The buffering inhibitor may also be a phospholipid. The phospholipidsmay be naturally occurring or non-naturally occurring phospholipids.Examples of phospholipids to be used as buffering inhibitors include butare not limited to phosphatidylcholine, phosphatidylethanolamine,phosphatidylglycerol, and phosphatidylserine. In some embodiments,phospholipids may be coated on the chemFET surface (or reaction chambersurface). Such coating may be covalent or non-covalent. In otherembodiments, the phospholipids exist in solution.

Still other methods relate to variations on sequencing-by-synthesismethods that increase the number of released hydrogen ions, againresulting in an increased signal (and signal to noise ratio). In somemethods, the number of hydrogen ions released per nucleotideincorporation are increased at least two-fold by combining a nucleotideincorporation event with a nucleotide excision event. An example of sucha process is a nick translation reaction in which a nucleotide isincorporated at a first position and another nucleotide is excised froma second, usually adjacent, position along a double stranded region of anucleic acid. The incorporation and excision each release one hydrogenion, and thus the coupling of the two events amplifies the number ofhydrogen ions per incorporation, thereby increasing signal.

Thus, in one aspect, there is provided a method comprising performing anick translation reaction along the length of a nicked, double strandednucleic acid, and detecting hydrogen ions released as a result of thenick translation reaction. In a related aspect, there is provided amethod comprising incorporating a first nucleotide at a first positionon a nucleic acid and excising a second nucleotide at a second positionon the nucleic acid, and detecting hydrogen ions released as a result ofnucleotide incorporation and excision. In another aspect, the there isprovided a method comprising incorporating a first known nucleotide at afirst position on a nucleic acid and excising a second nucleotide at asecond adjacent position on the nucleic acid, and detecting hydrogenions released as a result of nucleotide incorporation and excision. Instill another aspect, there is provided a method comprising sequentiallyexcising a nucleotide and incorporating another nucleotide at separatepositions along the length of a nicked, double stranded nucleic acid,and detecting hydrogen ions released from a combined nucleotide excisionand nucleotide incorporation, wherein released hydrogen ions areindicative of nucleotide incorporation and nucleotide excision. And inyet another aspect, there is provided a method comprising sequentiallycontacting a nicked, double stranded nucleic acid with each of fournucleotides in the presence of a polymerase, and detecting hydrogen ionsreleased following contact with each of the four nucleotides, whereinreleased hydrogen ions are indicative of nucleotide incorporation.

In another aspect, there is provided a method comprising detectingexcision of a first nucleotide and incorporation of a second knownnucleotide in a nicked, double stranded nucleic acid, in a solution incontact with or capacitively coupled to a ISFET. In one embodiment, thenicked, double stranded nucleic acid is a plurality of nicked, doublestranded nucleic acids.

In still another aspect, there is provided a method comprising detectingexcision of a nucleotide and incorporation of another nucleotide in aplurality of nicked double stranded nucleic acid present in a reactionchamber in contact with or capacitively coupled to an ISFET. In someembodiment, the reaction well is in a reaction chamber array and theISFET is in a ISFET array. In some embodiments, the ISFET arraycomprises 256 ISFET.

In another related aspect, a method is disclosed for improving signalfrom a sequencing-by-synthesis reaction comprising performing asequencing-by-synthesis reaction using a nick, double stranded templatenucleic acid, wherein at least one nucleotide incorporation event iscoupled to a nucleotide excision event, and wherein nucleotideincorporation events are detected by generation of a sequencing reactionbyproduct. In some embodiments, the sequencing reaction byproduct ishydrogen ions. In some embodiments, the hydrogen ions are detected by anISFET, which optionally may be present in an ISFET array.

The methods described herein may be performed in order to monitorreactions such as nick translation reactions, nucleotide incorporationsevents and/or nucleotide excision events. They may also be performed inorder to analyze a nucleic acid such as a template nucleic acid (whichmay be provided as a nicked, double stranded nucleic acid). Suchanalysis may include sequencing the template nucleic acid.

In some embodiments, released hydrogen ions are detected using an ISFETand/or an ISFET array. The ISFET array may comprise 256 ISFETs (i.e., itmay contain 256 or more ISFETs). In some embodiments, the ISFET array isoverlayed with a reaction chamber array.

In still other methods, the number of template nucleic acids used persensor, and optionally per reaction chamber, is increased. Since thesequencing-by-synthesis reactions contemplated by the inventiontypically occur simultaneously on a plurality of identical templatenucleic acids, increasing the number of templates increases the numberof sequencing byproduct (such as hydrogen ions) released persimultaneous nucleotide incorporation, thereby increasing signal thatcan be detected. Similarly, increasing the number of templatesimmobilized to an ISFET surface, as contemplated by some aspects of theinvention, increases the magnitude of the charge change observedfollowing nucleotide incorporation.

In some aspects described herein, increasing the concentration of thenucleic acids to be sequenced also serves to increase signal to noiseratio. Therefore in some instances decreasing the reaction volume (orthe reaction chamber volume) does not result in a decreased signal tonoise ratio, and can in fact result in an increased signal to noiseratio. In some instances, this may happen even if the total number ofnucleic acids being sequenced stays the same or is reduced.

Thus, in another aspect, the invention provides a method for sequencingnucleic acids comprising generating a plurality of template nucleicacids each comprising multiple, tandemly arranged, identical copies of atarget nucleic acid fragment, placing single template nucleic acids inreaction chambers of a reaction chamber array, and simultaneouslysequencing multiple template nucleic acids in reaction chambers of thereaction chamber array. In a related aspect, two or more templatenucleic acids which comprise multiple, tandemly arranged, identicalcopies of a target nucleic acid (or target nucleic acid fragment) areplaced in each reaction chamber. In this aspect, it is to be understoodthat the target nucleic acids (or target nucleic acid fragments) areidentical within a given chamber. The number of copies per template mayhowever vary, although preferably may also be similar or identical. Insome embodiments, sequencing multiple target nucleic acid fragmentscomprises detecting released hydrogen ions.

In some embodiments, the template nucleic acids are generated usingrolling circle amplification. In some embodiments, the template nucleicacids are attached to reaction chambers. In some embodiments, thereaction chamber array comprises 10², 10³, 10 ⁴, 10⁵, 10⁶ or 10⁷reaction chambers. In some embodiments, individual reaction chambers inthe reaction chamber array are in contact with or capacitively coupledto an chemFET. In some embodiments, the chemFET is in a chemFET array,and the chemFET array may optionally comprise 10², 10³, 10⁴, 10⁵, 10⁶ or10⁷ chemFETs. The chemFET and chemFET array may be an ISFET and an ISFETarray.

In another aspect, a method is provided for sequencing a nucleic acidcomprising generating a plurality of template nucleic acids eachcomprising multiple identical copies of a target nucleic acid (orfragment), placing single template nucleic acids in individual reactionchambers of a reaction chamber array, and sequencing multiple templatenucleic acids in reaction chambers of the reaction chamber array,wherein the single template nucleic acid has a cross-sectional areagreater than a cross-sectional area of the reaction chamber.

In one embodiment, single template nucleic acids are attached to singlereaction chambers in the reaction chamber array (i.e., only one templatenucleic acid is attached per reaction chamber). In one embodiment,single template nucleic acids are directly attached to single reactionchambers in the reaction chamber array. In some embodiments, the nucleicacid is not attached to the reaction chamber.

In still another aspect, an apparatus is provided that comprises anarray of chemFET each having a surface, and a plurality of templatenucleic acids each comprising multiple identical copies of a targetnucleic acid (or fragment), wherein single template nucleic acids arepresent on the surface of an individual chemFET. It is to be understoodthat the target nucleic acids within a template nucleic acid will beidentical but that those between template nucleic acids will typicallybe different from each other. In other words, each template in thisaspect is clonal. In one embodiment, single nucleic acids are attachedto the surface of individual chemFET. In one embodiment, the singlenucleic acids are directly attached to the surface of individualchemFET. In some embodiments, single nucleic acids are not attached tothe surface of individual chemFET.

Thus, it will be appreciated that in some embodiments, nucleic acids arepresent in a reaction chamber but are not attached to the surface of abead, although they may be attached or in contact with the chemFETsurface or a surface of the reaction chamber. Thus, in some embodiments,the reaction chambers comprise the nucleic acids to be sequenced even inthe absence of beads. In these. latter embodiments, the nucleic acidwithin a reaction chamber may comprise multiple (amplified) copies ofthe same nucleic acid to be sequenced. Single nucleic acids of this typeare deposited within single reaction chambers. These nucleic acids neednot be attached to the chemFET or reaction chamber surface.Alternatively, a plurality of amplified and physically separate nucleicacids may be present at or near a chemFET surface, and optionally withina reaction chamber.

The methods provided herein contemplate that the nucleic acids may beamplified while in contact with or near the chemFET surface, andoptionally within the reaction chamber, or that they may be amplifiedapart from either the chemFET and/or reaction chamber array and thendeposited onto a chemFET surface and/or into a reaction chamber.

Another aspect contemplates increasing the number of template nucleicacids present in or on nucleic acid-bearing beads. Thus, in one aspectthe invention provides a bead having a diameter less than 10 microns andhaving 1-5×10⁶ nucleic acids bound to its surface. In some embodiments,the bead has a diameter of about 1 micron, about 3 microns, about 5microns, or about 7 microns. In still other embodiments; the bead has adiameter of about 0.5 microns or about 0.1 microns. It will beunderstood that although such beads are characterized in some instancesaccording to their diameter, they need not be completely spherical inshape. In such instances, the diameter may refer to the diameteraveraged over a number of dimensions through the bead. In someembodiments, the bead comprises 1×10⁶ nucleic acids, 2×10⁶ nucleicacids, 3×10⁶ nucleic acids, or 4×10⁶ nucleic acids bound to its surface.In some embodiments, the nucleic acids are 5-50 nucleotides in length,10-50 nucleotides in length, or 20-50 nucleotides in length. In stillother embodiments, the nucleic acids are 50-1000 nucleotides in lengthor 1000-10000 nucleotides in length. The nucleic acids attached toand/or present in a bead are typically identical.

In some embodiments, the nucleic acids are synthetic nucleic acids(e.g., they have been synthesized using a nucleic acid synthesizer). Insome embodiments, the nucleic acids are amplification products.

In some embodiments, the nucleic acids are covalently bound to thesurface of the bead.

In some embodiments, the nucleic acids are bound to the surface of thebead with one or more non-nucleic acid polymers. In some embodiments,the non-nucleic acid polymers are polyethylene glycol (PEG) polymers.The PEG polymers may be of varying lengths. In some embodiments, one,some or all of the non-nucleic acid polymers comprises a plurality offunctional groups for nucleic acid binding. In some embodiments, thenon-nucleic acid polymers are dextran polymers and/or chitosan polymers.In some embodiments, the non-nucleic acid polymers include PEG polymersand dextran polymers. In some embodiments, the non-nucleic acid polymersinclude PEG polymers and chitosan polymers. The non-nucleic acidpolymers may be linear or branched.

In some embodiments, the nucleic acids are bound to a dendrimer that isbound to a bead. In some embodiments, the nucleic acids are bound to adendrimer that is bound to a PEG polymer.

In some embodiments, the nucleic acids are bound to the bead withself-assembling acrylamide monomers.

In various of these embodiments, the methods used to increase the numberof nucleic acids per bead provide no or minimal buffering to theenvironment.

In some embodiments, the bead is non-paramagnetic. In some embodiments,the bead has a density between 1-3 g/cm³. In some embodiments, the beadhas a density of about 2 g/cm³. In some embodiments, the bead is asilica bead. In some embodiments, the bead is a silica bead with anepoxide coat.

In a related aspect, a method is disclosed, comprising simultaneouslyincorporating known nucleotides into a plurality of the nucleic acidsimmobilized to and/or in a bead including but not limited to any of theforegoing beads. Immobilized as used herein includes but is not limitedto covalent or non-covalent attachment to a bead surface or interiorand/or simply physical retention within a porous bead, as described inmore detail herein. A plurality of these nucleic acids may be withoutlimitation 2-10², 2-10³, 2-10⁴, 2-10⁵, 2-10⁶, 2-2×10⁶, 2-3×10⁶, 2-4×10⁶or 2-5×10⁶ nucleic acids. Thus in some embodiments, the nucleotides areincorporated into at least 10⁶ nucleic acids, at least 2-10⁶ nucleicacids, at least 3-10⁶ nucleic acids, or at least 4-10⁶ nucleic acids. Itwill be understood that the maximum number of nucleic acids into whichnucleotides may be incorporated is the maximum number of nucleic acidsimmobilized to and/or in the bead. In some embodiments, the methodfurther comprises detecting nucleotide incorporation. In someembodiments, nucleotide incorporation is detected non-enzymatically. Insome embodiments, nucleotide incorporation is detected by detectingreleased hydrogen ions.

In some embodiments, the bead is in a reaction chamber, and optionallythe only bead in the reaction chamber. In some embodiments, the reactionchamber is in contact with or capacitively coupled to an ISFET. In someembodiments, the ISFET is in an ISFET array. In some embodiments, theISFET array comprises 10, 10², 10³, 10⁴, 10⁵ or 10⁶ ISFET.

In some embodiments, the bead has a diameter of less than 6 microns,less than 3 microns, or about 1 micron. The bead may have a diameter ofabout 1 micron up to about 7 microns, or about 1 micron up to about 3microns.

In some embodiments, the nucleic acids are self-priming template nucleicacids.

Thus, it will be understood that the invention contemplates sequencingof nucleic acids that are localized near a sensor such as an ISFETsensor (referred to herein as an ISFET), and optionally in a reactionchamber. The nucleic acids may be localized in a variety of waysincluding attachment to a solid support such as a bead surface, a beadinterior or some combination of bead surface and interior, as discussedabove. Typically, the bead is present in a reaction chamber, althoughthe methods may also be carried out in the absence of reaction chambers.The solid support may also be the sensor surface or a wall of a reactionchamber that is capacitively coupled to the sensor.

The localized nucleic acids are typically a plurality of identicalnucleic acids. The invention therefore further contemplatesamplification of nucleic acids while in contact with the chemFET (e.g.,ISFET) array (e.g., in the reaction chamber) followed by sequencing,with or without beads. The invention alternatively contemplatesintroducing a previously amplified population of nucleic acids toindividual sensors of a chemFET array, and optionally into individualreaction chambers, with or without beads.

Nucleic acids present in “porous” beads (or porous microparticles,porous microspheres or porous microcapsules, as the terms are usedinterchangeably herein) may be amplified and sequenced while individualbeads are in contact with individual chemFET sensors, optionally inindividual reaction chambers. Bridge amplification is one exemplarymethod for attaching identical nucleic acids onto a solid support suchas a bead surface, a chemFET surface, or a reaction chamber interiorsurface (e.g., a wall).

The nucleic acid-bearing beads used in various aspects and embodimentsof the invention include beads having nucleic acids attached to theirsurface, beads having nucleic acids in their internal core, or beadshaving nucleic acids attached to their surface and in their internalcore. Beads having nucleic acids in their internal core preferably havea porous surface that allows amplification and/or sequencing reagents tomove into and out of the bead but that retains the nucleic acids withinthe bead. Such beads therefore prevent the nucleic acids of interestfrom diffusing a significant distance away from the sensor, includingfor example diffusing out of a reaction chamber. The nucleic acidspresent in such beads may or may not be physically attached to the beadsbut they are nevertheless immobilized in the bead.

Accordingly, in another aspect, a disclosed method comprises detectinghydrogen ions as nucleotides are individually contacted with andincorporated into a plurality of identical nucleic acids in a reactionchamber in contact with or capacitively coupled to an ISFET, wherein thenucleic acids are present in a porous microparticle. In a relatedaspect, the invention provides a method comprising detecting hydrogenions as unblocked deoxyribonucleotides are individually contacted withand incorporated into a nucleic acid, in a reaction chamber in contactwith or capacitively coupled to an ISFET, wherein the nucleic acids arepresent in a porous microparticle. In some embodiments, the porousmicroparticle is hollow (i.e., it has a hollow core), while in otherembodiments it has a porous core.

Still another aspect of the disclosure provides a method for sequencingnucleic acids comprising generating a porous microparticle comprising asingle template nucleic acid (i.e., only a single template nucleic acidin the porous microparticle, initially) and polymerases, amplifying thesingle template nucleic acid in the porous microparticle, and sequencingamplified template nucleic acids in the porous microparticle.

In some embodiments, the amplified nucleic acids are sequenced in areaction chamber comprising a single microparticle (i.e., only a singlemicroparticle in the reaction chamber). The reaction chamber may bepresent in a reaction chamber array, and optionally the reaction chamberand/or the reaction chamber array may be in contact with or capacitivelycoupled respectively to a single ISFET or an ISFET array. In someembodiments, the reaction chambers in the reaction chamber array and/orthe ISFETs in the ISFET array have a center-to-center distance (betweenadjacent reaction chambers or ISFETs) ranging from about 1 micron toabout 10 microns.

In some embodiments, the method further comprises generating the singletemplate nucleic acids by fragmenting a larger nucleic acid (such as atarget nucleic acid).

In some embodiments, the amplified nucleic acids are sequenced withunlabeled nucleotide triphosphates and/or unblocked nucleotidetriphosphates.

In still another aspect, a disclosed method comprises providing in areaction chamber a single porous microparticle internally comprising aplurality of identical template nucleic acids, and sequencing theplurality of identical template nucleic acids simultaneously. As usedherein, “internally comprising” means that one, some or all of thenucleic acids are partially or completely present in the core of theporous microparticle. The plurality of identical template nucleic acidsmay be sequenced using a sequencing-by-synthesis method, as describedherein. The sequencing may comprise non-enzymatic detection ofnucleotide incorporation. The reaction chamber may be in contact with orcapacitively coupled to an ISFET, and/or it may be present in a reactionchamber array which is in contact with or capacitively coupled to anISFET array.

In another aspect, a method is provided for monitoring incorporation ofa nucleotide triphosphate into a nucleic acid comprising contacting aplurality of identical primers, a plurality of identical templatenucleic acids present in a porous microparticle, and a plurality ofidentical, known nucleotide triphosphates, in the presence of apolymerase, wherein the microparticle is present in a reaction chamberin contact with or capacitively coupled to a chemFET, and detecting asignal at the chemFET, wherein detection of the signal indicatesincorporation of the known nucleotide triphosphates to the primers.

In some embodiments, the signal results from release of a sequencingreaction byproduct such as PPi, Pi and/or hydrogen ions. In someembodiments, the chemFET is an ISFET. In some embodiments, the chemFETis in (or is provided in or as part of) a chemFET array. In someembodiments, the ISFET is in (or is provided in or as part of) an ISFETarray. In some embodiments, the chemFET or ISFET array comprises 10²,10³, 10⁴, 10⁵, 10⁶ or 10⁷ chemFETs or ISFETs respectively.

In some embodiments, the reaction chamber is in (or is provided in or aspart of) a reaction chamber array. In some embodiments, the reactionchamber array comprises 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ reactionchambers.

In some embodiments, the method further comprises generating theplurality of identical template nucleic acids by amplifying a singletemplate nucleic acid in the porous microparticle prior to contactingwith the plurality of identical primers. The plurality of identicaltemplate nucleic acids may be present in a concatemer or they may bephysically separate from each other.

In still another aspect, there is provided a method for sequencingnucleic acids comprising generating a plurality of template nucleicacids by fragmenting target nucleic acids, placing single templatenucleic acids in porous microparticles together with polymerases,amplifying the single template nucleic acids to generate a plurality ofidentical template nucleic acids in single porous microparticles,placing single porous microparticles in reaction chambers of a reactionchamber array, and simultaneously sequencing identical template nucleicacids in each of a plurality of porous microparticles.

In some embodiments, sequencing identical template nucleic acidscomprises detecting sequencing byproducts such as PPi, Pi and/orhydrogen ions released following nucleotide incorporation.

In some embodiments, the reaction chambers have a center-to-centerdistance of about 1 micron to about 10 microns. In some embodiments, thereaction chamber array comprises 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ reactionchambers.

In some embodiments, individual reaction chambers are in contact with orcapacitively coupled to individual chemFETs in a chemFET array,including individual ISFETs in an ISFET array. The chemFET or ISFETarray may comprise 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or more chemFETs orISFETs respectively. Adjacent sensors in these arrays may have acenter-to-center distance of about 1 micron to about 10 microns. Instill another aspect, the invention provides an apparatus comprising anISFET array and a plurality of porous microparticles each comprising aplurality of identical template nucleic acids, wherein single porousmicroparticles are in contact with single ISFETS within the array. Inone embodiment, the plurality of identical template nucleic acids aretandemly arranged in a single nucleic acid. In one embodiment, singleporous microparticles are present in single reaction chambers of areaction chamber array that is in contact with or capacitively coupledto the ISFET array. (We digress briefly on a definitional fine point.When a reaction chamber sits atop a dielectric that covers the floatingmetal gate of an ISFET, is that chamber in contact with the ISFET or isit capacitively coupled to the ISFET? This amounts to asking whether thedielectric is or is not part of the ISFET. We answer that it is part ofthe ISFET; otherwise, a direct electrical connection is being made to ametal gate and the would-be ISFET is simply a FET. However, we recognizethat the charge in the reaction chamber builds up on one side of thedielectric and forms one plate of a capacitor and which has as itssecond plate the floating gate metal layer; thus, we are alsocomfortable with the terminology stating that the reaction chamber iscapacitively coupled to the ISFET. The two alternatives thus areintended to mean the same thing.)

Some aspects of the invention involve detection of charge bound to thechemFET (including an ISFET) surface. Such detection can be used aloneor together with detection of soluble analytes (such as hydrogen ions)to detect an event such as for example a nucleotide incorporation event.Thus, as an example, a sequencing-by-synthesis reaction may occur usinga template nucleic acid that is immobilized to a chemFET surface.Nucleotide incorporation into the newly synthesized strand results in anaddition of negative charge to the nucleic acid and this change can besensed by the chemFET. Nucleotide incorporation also results in therelease of PPi, and subsequently a hydrogen ion, which can also besensed by the chemFET. Some embodiments involve sequencing such surfaceimmobilized templates in the presence of sufficient buffer to quench (ormask) any released hydrogen ions, thereby tracking a signal that resultsonly from addition of negative charge to the surface as an indicator ofnucleotide incorporation. In some embodiments, the method is carried outin the absence of a buffer. Thus, in another aspect, the inventionprovides a method for sequencing a nucleic acid comprising amplifying asingle template nucleic acid in a reaction chamber in contact with orcapacitively coupled to an ISFET, wherein amplified template nucleicacids are attached to the reaction chamber, and sequencing amplifiedtemplate nucleic acids in the reaction chamber.

In some embodiments, the amplified template nucleic acids are attachedto the surface of the ISFET. In some embodiments, the single templatenucleic acid is attached to a surface of the ISFET prior toamplification. In some embodiments, the single template nucleic acid isamplified in solution and the amplified template nucleic acids arehybridized to primers immobilized on a surface of the ISFET.

In some embodiments, amplifying comprises amplifying by rolling circleamplification, and the amplified template nucleic acids are concatemersof the template nucleic acid.

In some embodiments, sequencing comprises detecting incorporation of aknown nucleotide by an increase in negative charge of the amplifiedtemplate nucleic acids.

In some embodiments, the amplified template nucleic acids areself-priming.

In still another aspect, a method is provided, comprising contacting aknown nucleotide to a complex comprising a template nucleic acid and asequencing primer, wherein the complex is immobilized on a surface of anISFET, and detecting incorporation of the known nucleotide to thecomplex by detecting an increase in negative charge of the complex,wherein the ISFET is in an array, and optionally wherein the arraycomprises 256 ISFETs.

In some embodiments, the template nucleic acid is present in (orprovided as) a concatemer of template nucleic acids. In someembodiments, the concatemer comprises 100-1000 copies of the templatenucleic acid. In some embodiments, the template nucleic acid iscovalently bound to the surface of the ISFET. In some embodiments, thesequencing primer is covalently bound to the surface of the ISFET.

In some embodiments, the ISFET is overlayed with a reaction chamber, andoptionally the reaction chamber is in an array. In some embodiments, thereaction chamber contains a buffered solution.

In some embodiments, the complex is a plurality of complexes. In someembodiments, the complexes are identical. In some embodiments, theplurality of complexes is equal to or less than 10⁶ complexes, equal toor less than 10⁵ complexes, equal to or less than 10⁴ complexes, orequal to or less than 10³ complexes.

In yet another aspect, a method comprises contacting a known nucleotideto a self-priming template nucleic acid that is immobilized on a surfaceof an ISFET, and detecting incorporation of the known nucleotide to theself-priming template nucleic acid by detecting an increase in negativecharge of the nucleic acid.

In some embodiments, the ISFET is in an ISFET array. The ISFET array maycomprise 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ ISFETs.

In some embodiments, the template nucleic acid is in a reaction chamberin contact with or capacitively coupled to the ISFET. In someembodiments, the reaction chamber is in a reaction chamber array. Insome embodiments, the reaction chamber array comprises 10², 10³, 10⁴,10⁵, 10⁶ or 10⁷ reaction chambers.

In some embodiments, the nucleic acid is in a buffer. Thus, in someembodiments, signal at the ISFET results solely from a change in chargeof the nucleic acid rather than from released hydrogen ions.

Thus, it is to be understood that various aspects and embodiments of theinvention relate generally to large scale FET arrays for measuring oneor more analytes or for measuring charge bound to the chemFET surface.It will be appreciated that chemFETs and more particularly ISFETs may beused to detect analytes and/or charge. An ISFET, as discussed above, isa particular type of chemFET that is configured for ion detection suchas hydrogen ion (or proton) detection. Other types of chemFETscontemplated by the present disclosure include enzyme FETs (EnFETs)which employ enzymes to detect analytes. It should be appreciated,however, that the present disclosure is not limited to ISFETs andEnFETs, but more generally relates to any FET that is configured forsome type of chemical sensitivity. As used herein, chemical sensitivitybroadly encompasses sensitivity to any molecule of interest, includingwithout limitation organic, inorganic, naturally occurring,non-naturally occurring, chemical and biological compounds, such asions, small molecules, polymers such as nucleic acids, proteins,peptides, polysaccharides, and the like.

Various embodiments described herein employ large scale chemFET arraysin the analysis of chemical or biological samples and/or reactions.Chemical or biological samples are typically liquid (or are dissolved ina liquid) and of small volume, to facilitate high-speed, high-densitydetermination of analyte (e.g., ion or other constituent) presenceand/or concentration, or other analyte measurements.

For example, some embodiments involve a “very large scale”two-dimensional chemFET sensor array (e.g., greater than 256 sensors),in which one or more chemFET-containing elements or “pixels”constituting the sensors of such an array are configured to monitor oneor more independent biological or chemical reactions or events occurringin proximity to the pixels of the array. It will be understood that sucharrays may comprise any number of individual sensors and that theinvention is not to be limited in this regard. In some exemplaryimplementations, the array may be coupled to one or more microfluidicsstructures that form one or more reaction chambers, or “wells” or“microwells,” (as the terms are used interchangeably herein) overindividual sensors or groups of sensors of the array, and an apparatusthat delivers analyte samples (i.e., analyte solutions) to the wellsand/or removes them from the wells between measurements. Even whenmicrowells are not employed, the sensor array may be coupled to one ormore microfluidics structures for the delivery of one or more samples tothe pixels and for removal of sample between measurements. Inassociation with the microfluidics, unique reference electrodes andtheir coupling to the flow cell are also provided by the invention.

Accordingly, disclosed herein are various microfluidic structures whichmay be employed to flow analytes and, where appropriate, other agentsuseful in for example the detection and measurement of analytes to andfrom the reaction chambers or pixels, the methods of manufacture of thearray of reaction chambers, methods and structures for coupling thearrayed reaction chambers with arrayed pixels, and methods and apparatusfor loading the reaction chambers with sample to be analyzed, includingfor example loading the wells with nucleic acids for example when theapparatus is used for nucleic acid (e.g., DNA) sequencing or relatedanalysis, and uses thereof, as will be discussed in greater detailherein. In various aspects of the invention, an analyte that isbyproduct of a nucleic acid synthesis reaction is detected. Such abyproduct can be monitored as the readout of a sequencing-by-synthesismethod. One particularly important byproduct is hydrogen ions which arereleased upon addition or incorporation of a deoxynucleotidetriphosphate (also referred to herein as a nucleotide or a dNTP) to the3′ end of a nucleic acid (such as a sequencing primer). Nucleotideincorporation releases inorganic pyrophosphate (PPi) which may behydrolyzed to orthophosphate (Pi) and free hydrogen ion (H⁺) in thepresence of water (and optionally and far more rapidly in the presenceof pyrophosphatase). As a result, nucleotide incorporation, and thus asequencing-by-synthesis reaction, can be monitored by detecting PPi, Piand/or H⁺. Conventionally, PPi has not been detected or measured bychemFETs. Instead, optically based sequencing-by-synthesis methods havedetected PPi via its sulfurylase-mediated conversion to adenosinetriphosphate (ATP), and then luciferase-mediated conversion of luciferinto oxyluciferin in the presence of the previously generated ATP, withconcomitant release of light. Such detection is referred to herein as“enzymatic” detection (e.g., of released PPi or of nucleotideincorporation).

As mentioned above, in some aspects the invention provides methods fordetecting nucleotide incorporation (optionally in conjunction withnucleotide excision) using non-enzymatic methods. As used herein,non-enzymatic detection of nucleotide incorporation is detection thatdoes not require an enzyme to detect the incorporation event orbyproducts thereof. Non-enzymatic detection however does not exclude theuse of enzymes to incorporate nucleotides or, in some instances, toexcise nucleotides, thereby generating the event that is being detected.An example of non-enzymatic detection of nucleotide incorporation is adetection method that does not require conversion of PPi to ATP.Non-enzymatic detection methods may employ mixtures of polymerases fornucleotide incorporation, or they may employ enzymes that may enhance asignal (e.g., pyrophosphatase in order to enhance conversion of PPi toPi), enzymes that reduce misincorporations (e.g., apyrase in order toremove unincorporated nucleotides), and/or enzymes that removenucleotides in conjunction with incorporation of other nucleotides,among others.

Thus, in some aspects the instant invention contemplates and providesmethods for monitoring nucleic acid sequencing reactions and thusdetermining the nucleotide sequence of nucleic acids by detecting H+ (orchanges in pH), PPi (or Pi, or changes in either) in the absence orpresence of PPi (or Pi) specific receptors, alone or in some combinationthereof.

In other aspects, other biological or chemical reactions may bemonitored, and the chemFET arrays may be specifically configured tomeasure hydrogen ions and/or one or more other analytes that providerelevant information relating to the occurrence and/or progress of aparticular biological or chemical process of interest.

With respect to analyte detection and measurement, it should beappreciated that in various embodiments discussed in greater detailbelow, one or more analytes measured by a chemFET array according to thepresent disclosure may include any of a variety of biological orchemical substances that provide relevant information regarding abiological or chemical process (e.g., binding events such ashybridization of nucleic acids to each other, antigen-antibody binding,receptor-ligand binding, enzyme-inhibitor binding, enzyme-substratebinding, enzyme-agonist binding, enzyme-antagonist binding, and thelike). In some aspects, the ability to measure absolute or relative aswell as static and/or dynamic levels and/or concentrations of one ormore analytes, in addition to merely determining the presence or absenceof an analyte, provides valuable information in connection withbiological and chemical processes. In other aspects, mere determinationof the presence or absence of an analyte or analytes of interest mayprovide valuable information and may be sufficient.

A chemFET array according to various inventive embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes, and in other embodiments different chemFETs of a given arraymay be configured for sensitivity to different analytes. For example, inone embodiment, one or more sensors (pixels) of the array may include afirst type of chemFET configured to be sensitive to a first analyte, andone or more other sensors of the array may include a second type ofchemFET configured to be sensitive to a second analyte different fromthe first analyte. In one embodiment, the first and second analytes maybe related to each other. As an example, the first and second analytesmay be byproducts of the same biological or chemical reaction/processand therefore they may be detected concurrently to confirm theoccurrence of a reaction (or lack thereof). Such redundancy ispreferable in some analyte detection methods. Of course, it should beappreciated that more than two different types of chemFETs may beemployed in any given array to detect and/or measure different types ofanalytes, and optionally to monitor biological or chemical processessuch as binding events. In general, it should be appreciated in any ofthe embodiments of sensor arrays discussed herein that a given sensorarray may be “homogeneous” and thereby consist of chemFETs ofsubstantially similar or identical type that detect and/or measure thesame analyte (e.g., pH or other ion concentration), or a sensor arraymay be “heterogeneous” and include chemFETs of different types to detectand/or measure different analytes. In another embodiment, the sensors inan array may be configured to detect and/or measure a single type (orclass) of analyte even though the species of that type (or class)detected and/or measured may be different between sensors. As anexample, all the sensors in an array may be configured to detect and/ormeasure nucleic acids, but each sensor detects and/or measures adifferent nucleic acid.

Aspects of the invention provide specific improvements to the ISFETarray design of Milgrew et al. discussed above in connection with FIGS.1-7, as well as other conventional ISFET array designs, so as tosignificantly reduce pixel size, and thereby increase the number ofpixels of a chemFET array for a given semiconductor die size (i.e.,increase pixel density). In various embodiments, this increase in pixeldensity is accomplished while at the same time increasing thesignal-to-noise ratio of output signals corresponding to monitoredbiological and chemical processes, and the speed with which such outputsignals may be read from the array. In particular, by relaxingrequirements for chemFET linearity and focusing on a more limitedmeasurement output signal range (e.g., output signals corresponding to apH range of from approximately 7 to 9 or smaller, rather than 1 to 14,as well as output signals that do not necessarily relate significantlyto pH), individual pixel complexity and size may be significantlyreduced, thereby facilitating the realization of very large scale densechemFET arrays. Alternative less complex approaches to pixel selectionin an chemFET array (e.g., alternatives to the row and column decoderapproach employed in the design of Milgrew et al. as shown in FIG. 7,whose complexity scales with array size), as well as various dataprocessing techniques involving ISFET response modeling and dataextrapolation based on such modeling, facilitate rapid acquisition ofdata from significantly large and dense arrays.

In various aspects, the chemFET arrays may be fabricated usingconventional CMOS (or biCMOS or other suitable) processing technologies,and are particularly configured to facilitate the rapid acquisition ofdata from the entire array (scanning all of the pixels to obtaincorresponding pixel output signals).

Various techniques employed in a conventional CMOS fabrication process,as well as various post-fabrication processing steps (wafer handling,cleaning, dicing, packaging, etc.), may in some instances adverselyaffect performance of the resulting chemFET array. For example, withreference again to FIG. 1, one potential issue relates to trapped chargethat may be induced in the gate oxide 65 during etching of metalsassociated with the floating gate structure 70, and how such trappedcharge may affect chemFET threshold voltage V_(TH). Another potentialissue relates to the density/porosity of the chemFET passivation layer(e.g., see ISFET passivation layer 72 in FIG. 1) resulting fromlow-temperature material deposition processes commonly employed inaluminum metal-based CMOS fabrication. While such low-temperatureprocesses generally provide an adequate passivation layer forconventional CMOS devices, they may result in a somewhat low-density andporous passivation layer which may be potentially problematic forchemFETs in contact with an analyte solution; in particular, alow-density porous passivation layer over time may absorb and becomesaturated with analytes or other substances in the solution, which mayin turn cause an undesirable time-varying drift in the chemFETsthreshold voltage V_(TH). This phenomenon may in turn impede accuratemeasurements of one or more particular analytes of interest. In view ofthe foregoing, other inventive embodiments disclosed herein relate tomethods and apparatuses which mitigate potentially adverse effects onchemFET performance that may arise from various aspects of fabricationand post-fabrication processing/handling of chemFET arrays.

Accordingly, one embodiment is directed to an apparatus, comprising anarray of CMOS-fabricated sensors, each sensor comprising onechemically-sensitive field effect transistor (chemFET) and occupying anarea on a surface of the array of 10 μm² or less, 9 μm² or less, 8 μm²or less, 7 μm² or less, 6 μm² or less, 5 μm² or less, 4 μm² or less 3μm² or less, or 2 μm² or less.

Another embodiment is directed to a sensor array, comprising atwo-dimensional array of electronic sensors including at least 512 rowsand at least 512 columns of the electronic sensors, each sensorcomprising one chemically-sensitive field effect transistor (chemFET)configured to provide at least one output signal representing a presenceand/or concentration of an analyte proximate to a surface of thetwo-dimensional array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising one chemically-sensitivefield effect transistor (chemFET). The array of CMOS-fabricated sensorsincludes more than 256 sensors, and a collection of chemFET outputsignals from all chemFETs of the array constitutes a frame of data. Theapparatus further comprises control circuitry coupled to the array andconfigured to generate at least one array output signal to providemultiple frames of data from the array at a frame rate of at least 1frame per second. In one aspect, the frame rate may be at least 10frames per second. In another aspect, the frame rate may be at least 20frames per second. In yet other aspects, the frame rate may be at least30, 40, 50, 70 or up to 100 frames per second.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor consisting of three field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET). Another embodiment is directed to an apparatus,comprising an array of electronic sensors, each sensor comprising threeor fewer field effect transistors (FETs), wherein the three or fewerFETs includes one chemically-sensitive field effect transistor(chemFET).

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor comprising a plurality of field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET), and a plurality of electrical conductorselectrically connected to the plurality of FETs, wherein the pluralityof FETs are arranged such that the plurality of electrical conductorsincludes no more than four conductors traversing an area occupied byeach sensor and interconnecting multiple sensors of the array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a plurality of fieldeffect transistors (FETs) including one chemically-sensitive fieldeffect transistor (chemFET), wherein all of the FETs in each sensor areof a same channel type and are implemented in a single semiconductorregion of an array substrate.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one and in someinstances at least two output signals representing a presence and/or aconcentration of an analyte proximate to a surface of the array. Foreach column of the plurality of columns, the array further comprisescolumn circuitry configured to provide a constant drain current and aconstant drain-to-source voltage to respective chemFETs in the column,the column circuitry including two operational amplifiers and adiode-connected FET arranged in a Kelvin bridge configuration with therespective chemFETs to provide the constant drain-to-source voltage.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one output signaland in some instances at least two output signals representing aconcentration of ions in a solution proximate to a surface of the array.The array further comprises at least one row select shift register toenable respective rows of the plurality of rows, and at least one columnselect shift register to acquire chemFET output signals from respectivecolumns of the plurality of columns.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.The array includes a two-dimensional array of at least 512 rows and atleast 512 columns of the CMOS-fabricated sensors. Each sensor consistsof three field effect transistors (FETs) including the chemFET, and eachsensor includes a plurality of electrical conductors electricallyconnected to the three FETs. The three FETs are arranged such that theplurality of electrical conductors includes no more than four conductorstraversing an area occupied by each sensor and interconnecting multiplesensors of the array. All of the FETs in each sensor are of a samechannel type and implemented in a single semiconductor region of anarray substrate. A collection of chemFET output signals from allchemFETs of the array constitutes a frame of data. The apparatus furthercomprises control circuitry coupled to the array and configured togenerate at least one array output signal to provide multiple frames ofdata from the array at a frame rate of at least 20 frames per second.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The method comprises: A) dicing asemiconductor wafer including the array to form at least one dicedportion including the array; and B) performing a forming gas anneal onthe at least one diced portion.

Another embodiment is directed to a method for manufacturing an array ofchemFETs. The method comprises fabricating an array of chemFETs;depositing on the array a dielectric material; applying a forming gasanneal to the array before a dicing step; dicing the array; and applyinga forming gas anneal after the dicing step. The method may furthercomprise testing the semiconductor wafer between one or more depositionsteps.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors. Each sensor comprises a chemically-sensitivefield effect transistor (chemFET) having a chemically-sensitivepassivation layer of silicon nitride and/or silicon oxynitride depositedvia plasma enhanced chemical vapor deposition (PECVD). The methodcomprises depositing at least one additional passivation material on thechemically-sensitive passivation layer so as to reduce a porosity and/orincrease a density of the passivation layer.

Various aspects or embodiments of the invention involve an apparatuscomprising an array of chemFET sensors overlayed with an array ofreaction chambers wherein the bottom of a reaction chamber is in contactwith (or capacitively coupled to) a chemFET sensor. In some embodiments,each reaction chamber bottom is in contact with a chemFET sensor, andpreferably with a separate chemFET sensor. In some embodiments, lessthan all reaction chamber bottoms are in contact with a chemFET sensor.In some embodiments, each sensor in the array is in contact with areaction chamber. In other embodiments, less than all sensors are incontact with a reaction chamber. The sensor (and/or reaction chamber)array may be comprised of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 60, 80, 90, 100, 200, 300, 400, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, or more chemFET sensors (and/or reaction chambers). As used herein,it is intended that an array that comprises, as an example, 256 sensorsor reaction chambers will contain 256 or more (i.e., at least 256)sensors or reaction chambers. It is further intended that aspects andembodiments described herein that “comprise” elements and/or steps alsofully support and embrace aspects and embodiments that “consist of” or“consist essentially of” such elements and/or steps.

Various aspects and embodiments of the invention involve sensors (and/orreaction chambers) within an array that are spaced apart from each otherat a center-to-center distance or spacing (or “pitch”, as the terms areused interchangeably herein) that is in the range of 1-50 microns, 1-40microns, 1-30 microns, 1-20 microns, 1-10 microns, or 5-microns,including equal to or less than about 9 microns, or equal to or lessthan about 5.1 microns, or 1-5 microns including equal to or less thanabout 2.8 microns. The center-to-center distance between adjacentreaction chambers in a reaction chamber array may be about 1-9 microns,or about 2-9 microns, or about 1 microns, about 2 microns, about 3microns, about 4 microns, about 5 microns, about 6 microns, about 7microns, about 8 microns, or about 9 microns.

In some embodiments, the reaction chamber has a volume of equal to orless than about 1 picoliter (pL), including less than 0.5 pL, less than0.1 pL, less than 0.05 pL, less than 0.01 pL; less than 0.005 pL.

The reaction chambers may have a square cross section, for example, attheir base or bottom. Examples include an 8 μm by 8 μm cross section, a4 μm by 4 μm cross section, or a 1.5 μm by 1.5 μm cross section.Alternatively, they may have a rectangular cross section, for example,at their base or bottom. Examples include an 8 μm by 12 μm crosssection, a 4 μm by 6 μm cross section, or a 1.5 μm by 2.25 μm crosssection.

In some embodiments, a reaction chamber comprises a single templatenucleic acid or a single bead. In these instances, it is to beunderstood that such reaction chambers have only one template nucleicacid or only one bead, although they may contain other elements. Such“single nucleic acids” however may be later amplified in order to giverise to a plurality of identical nucleic acids. Similarly, in someembodiments, a single template nucleic acid may be a concatemer and thusmay contain multiple copies of a starting nucleic acid such as astarting template nucleic acid or a target nucleic acid fragment. Asused herein, a plurality is two or more.

In some embodiments, a reaction chamber comprises a plurality ofidentical nucleic acids. In some embodiments, the identical nucleicacids are attached (e.g., covalently) to a bead within the well. Inother embodiments, the identical nucleic acids are attached (e.g.,covalently) to a surface in the reaction chamber such as but not limitedto the chemFET surface (or typically at the bottom of the reactionchamber). The plurality of nucleic acids can be 2-10, 2-10², 2-10³,2-10⁴, 2-10⁵, 2-10⁶, or more. In some embodiments, the plurality ofnucleic acids can be 2 through to 2 million, 2 through to 3 million, 2through to 4 million, 2 through to 5 million, or more. As used herein, atemplate nucleic acid may contain a single template or it may contain aplurality of templates (e.g., in the case of a concatemer, whether ornot in the context of a DNA “nanoball”). Such concatemers may include10, 50, 100, 500, 1000, or more copies of the template nucleic acid.When such concatemers are used, they may exist in a reaction well, orotherwise be in close proximity to the chemFET surface, in the absenceor presence of a bead. That is, the concatemers may be presentindependently of beads, and they may or may not be themselves covalentlyor non-covalently attached to the chemFET surface. Sequencing of suchnucleic acids may be via detection of released hydrogen ions and/ordetection of addition of negative charge to the chemFET surfacefollowing nucleotide incorporations events.

Other aspects of the invention relate to methods for monitoring nucleicacid synthesis reactions, including but not limited to those integral tosequencing-by-synthesis methods. Thus, various aspects of the inventionprovide methods for monitoring nucleic acid synthesis reactions, methodsfor determining or monitoring nucleotide incorporation into a nucleicacid, and the like, optionally in the presence of nucleotide excision asmay occur for example in a nick translation reaction. These methods arecarried out in some important embodiments in a pH sensitive environment(i.e., an environment in which pH and pH changes can be detected).

Various methods provided herein rely on sequencing a nucleic acid bycontacting a plurality of the nucleic acids sequentially to a knownorder of different nucleotides (e.g., dATP, dCTP, dGTP, and dTTP), anddetecting an electrical output that results if the nucleotide isincorporated. Some methods employ a primed template nucleic acid andincorporate nucleotides into a sequencing primer based oncomplementarity with the template nucleic acid.

Thus, some aspects of the invention provide methods for sequencing anucleic acid comprising sequencing a plurality of identical templatenucleic acids in a reaction chamber in contact with a chemFET, in anarray which comprises at least 3 (and up to millions) of such assembliesof reaction chambers and chemFETs.

Some methods involve sequencing individually amplified fragmentednucleic acids using a chemFET array, optionally overlayed with areaction chamber array. In various embodiments, the chemFET arraycomprises at least 500 chemFETs, at least 100,000 chemFETs, at least 1million chemFETs, or more. In some embodiments, the plurality offragmented nucleic acids is individually amplified using a water in oilemulsion amplification method.

Some methods involve disposing (e.g., placing or positioning) aplurality of identical template nucleic acids into a reaction chamber(or well) that is in contact with or capacitively coupled to a chemFET,wherein the template nucleic acids are individually hybridized tosequencing primers or are self-priming (thereby forming atemplate/primer hybrid), synthesizing a new nucleic acid strand (orextending the sequencing primer) by incorporating one or more knownnucleotide triphosphates sequentially at the 3′ end of the sequencingprimer in the presence of a polymerase, and detecting the incorporationof the one or more known nucleotide triphosphates by a change in voltageand/or current at the chemFET. The chemFET is preferably one sensor in achemFET array and the reaction chamber is preferably one chamber in areaction chamber array. The template nucleic acids between reactionchambers may differ but those within a reaction chamber are preferablyidentical. Thus it will be clear that the invention contemplatesperforming a plurality of sequencing reactions simultaneously within areaction chamber and if in the context of an array within the pluralityof reaction chambers in the array.

The above-noted methods may be carried out on templates that areimmobilized (e.g., covalently) to a bead located within the reactionchamber or on templates that are immobilized (e.g., covalently) to asurface inside the reaction chamber including the chemFET surface.Nucleotide incorporation can then be detected by an increase in therelease of hydrogen ions into the solution and ultimately in contactwith the chemFET surface and/or by an increase in the negative charge atthe chemFET surface.

Various embodiments may be embraced in the various foregoing aspects ofthe invention and these are recited below once for convenience andbrevity.

It is to be understood that although various of the foregoing aspectsand embodiments of the invention recite hybridization (or binding) of asequencing primer to a template, the invention also contemplates the useof template nucleic acids that hybridize to themselves (i.e.,intramolecularly) thereby giving rise to free 3′ ends onto whichnucleotide triphosphates may be incorporated. Such templates, referredto herein as self-priming templates, may be used in any of the foregoingmethods.

Similarly, the invention equally contemplates the use of double strandedtemplates that are engineered to have particular sequences at their freeends that can be acted upon by nicking enzymes such as nickases. In thisway, the polymerase incorporates nucleotide triphosphates at the nickedsite. In these instances, there is no requirement for a separatesequencing primer. The double stranded template may compriseribonucleotide (i.e., RNA) bases including for example uracils which areacted upon by different enzymes to create a nick in the template fromwhich sequencing may begin. It is to be understood that such methods arestill considered “non-enzymatic” as intended herein since the detectionof nucleotide incorporation (via detection of a released product orbyproduct of the incorporation reaction or by detection of an increasedcharge at the chemFET surface) does not rely on an enzyme, even thoughthe nucleotide incorporation event typically does.

In various embodiments, the incorporated nucleotide triphosphate isknown. In various embodiments, the nucleotide triphosphate is aplurality of identical nucleotide triphosphates, the template is aplurality of templates, the hybrids are a plurality of hybrids, and thepolymerase is a plurality of polymerases. The polymerase may be aplurality of polymerases that are not identical and rather may becomprised of 2, 3, or more types of polymerases. In some instances, amixture of two polymerases may be used with one having suitableprocessivity and the other having suitable rate of incorporation. Theratio of the different polymerases can vary. Similarly, the primer,template or hybrid may be a plurality of primers, templates, or hybridsrespectively that may not be identical to each other, provided that anyprimer, template or hybrid in a single reaction chamber, attached to asingle capture bead or to another solid support such as a chemFETsurface in the same reaction chamber are identical to each other. Insome instances, the primers are identical between reaction chambers.

In some embodiments, the incorporation of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, or at least 100 nucleotide triphosphates is detected.In other embodiments, the incorporation of 100-500 25-750, 500-1000, or10-1000 nucleotide triphosphates is detected.

In some embodiments, the reaction chamber comprises a plurality ofpacking beads. In some embodiments, the reaction chamber lacks packingbeads.

In some embodiments, the reaction chamber comprises a solublenon-nucleic acid polymer. In some embodiments, the detecting step occursin the presence of a soluble non-nucleic acid polymer. In someembodiments, the soluble non-nucleic acid polymer is polyethyleneglycol, or PEA, or a dextran, or an acrylamide, or a cellulose (e.g.,methyl cellulose). In some embodiments, the non-nucleic acid polymersuch as polyethylene glycol is attached to the single bead. In someembodiments, the non-nucleic acid polymer is attached to one or more (orall) sides of a reaction chamber, except in some instances the bottom ofthe reaction chamber which is the FET surface. In some embodiments, thenon-nucleic acid polymer is biotinylated such as but not limited tobiotinylated polyethylene glycol.

In some embodiments, the method is carried out at a pH of about 6-9.5,or at about 6-9, or at about 7-9, or at about 8.5 to 9.5, or at about 9.The pH range in some instances is dictated by the polymerase (and/orother enzyme) being used in the method.

In some important embodiments, the synthesizing and/or detecting step iscarried out in a weak buffer. In some embodiments, the weak buffercomprises Tris-HCl, boric acid or borate buffer, acetate, morpholine,citric acid, carbonic acid, or phosphoric acid as a buffering agent. Insome embodiments, the synthesizing and/or detecting step is carried outin an aqueous solution that lacks buffer.

In some embodiments, the synthesizing and/or detecting step is carriedout in about 1 mM Tris-HCl. In some embodiments, the synthesizing and/ordetecting step is carried out in less than 1 mM Tris-HCl. In someembodiments, the synthesizing and/or detecting step is carried out inabout 0.9 mM Tris-HCl, about 0.8 mM Tris-HCl, about 0.7 mM Tris-HCl,about 0.6 mM Tris-HCl, about 0.5 mM Tris-HCl, about 0.4 mM Tris-HCl,about 0.3 mM Tris-HCl, or about 0.2 mM Tris-HCl.

In some embodiments, the synthesizing and/or detecting step is carriedout in about 1 mM borate buffer. In some embodiments, the synthesizingand/or detecting step is carried out in less than 1 mM borate buffer. Insome embodiments, the synthesizing and/or detecting step is carried outin about 0.9 mM borate buffer, about 0.8 mM borate buffer, about 0.7 mMborate buffer, about 0.6 mM borate buffer, about 0.5 mM borate buffer,about 0.4 mM borate buffer, about 0.3 mM borate buffer, or about 0.2 mMborate buffer.

In various embodiments, the nucleotide triphosphates are unblocked. Asused herein, an unblocked nucleotide triphosphate is a nucleotidetriphosphate with an unmodified end that can be incorporated into anucleic acid (at its 3′ end) and once it is incorporated can be attachedto the following nucleotide triphosphate being incorporated. BlockeddNTP in contrast either cannot be added to a nucleic acid or theirincorporation into a nucleic acid prevents any further nucleotideincorporation and any further extension of that nucleic acid. In variousembodiments, the nucleotide triphosphates are deoxynucleotidetriphosphates (dNTPs).

In various embodiments, the chemFET comprises a silicon nitridepassivation layer. The passivation layer may or may not be bound to anucleic acid such as a template nucleic acid or a concatemer of templatenucleic acids.

In some embodiments, the nucleotide triphosphates are pre-soaked in Mg²⁺(e.g., in the presence of MgCl₂) or Mn²⁺ (e.g., in the presence ofMnCl₂). In some embodiments, the polymerase is pre-soaked in Mg²⁺ (e.g.,in the presence of MgCl₂) or Mn²⁺ (e.g., in the presence of MnCl₂).

In some embodiments, the method is carried out in a reaction chambercomprising a single capture bead, wherein a ratio of reaction chamberwidth to single capture bead diameter is at least 0.7, at least 0.8, orat least 0.9.

In some embodiments, the polymerase is free in solution. In someembodiments, the polymerase is immobilized to a bead. In someembodiments, the polymerase is immobilized to a capture bead. In someembodiments, the template nucleic acids are attached to capture beads.In some embodiments, the template nucleic acids are attached to thechemFET surface or another wall inside the reaction chamber.

A number of aspects of the disclosed apparatus relate to improvingperformance by, for example, improving the signal-to-noise ratio ofindividual ISFET-based pixels as well as arrays of such pixels.

One aspect involves One aspect involves over-coating (i.e.,“passivating”) the sidewalls (typically formed of TEOS-oxide or anothersuitable material, as above-described) and sensor surface at the bottomof the microwells with various metal oxide or like materials, to improvetheir surface chemistry (i.e., make the sidewalls less reactive) andelectrical properties.

Another aspect is forming ISFETs with a very thin dielectric coating onthe floating gate electrode.

Yet another aspect is forming a combined ISFET and microwell structurewherein the surface area for charge collection at the floating gate isincreased by employing a metallization on the microwell sidewalls.

Still a further aspect is employing modified array and pixels designs toreduce noise sources, including charge injection into the electrolyte.In part, these designs include the use of active pixels having currentsources configured to reduce ISFET terminal voltage fluctuations.

Yet another aspect is providing a more reliable way to introduce astable reference potential into a flow cell having a solution flowingtherethrough, such that the reference potential will be substantiallyinsensitive to spatial variations in fluid composition and pH.

A further aspect is an improved mechanism for multiplexing fluid flowsinto the flow cell, whereby switching of fluids is simplified andinstead of multiplexing multiple reagents at the location of valves usedto control their flow, reagents are multiplexed downstream with apassive micro-fluidic multiplexer circuit that acts as a kind of union.Diffusion-transported effluent is minimized from reagent inputs otherthan the one currently being used. Laminar flow and/or fluid resistanceelements cause diffuse effluent to be discarded to a waste location.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead being placed upon generallyillustrating the various concepts discussed herein.

FIG. 1 illustrates a cross-section of a p-type (p-channel) ion-sensitivefield effect transistor (ISFET) fabricated using a conventional CMOSprocess.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET shown in FIG. 1.

FIG. 2A illustrates an exemplary ISFET transient response to astep-change in ion concentration of an analyte.

FIG. 3 illustrates one column of a two-dimensional ISFET array based onthe ISFET shown in FIG. 1.

FIG. 4 illustrates a transmission gate including a p-channel MOSFET andan n-channel MOSFET that is employed in each pixel of the array columnshown in FIG. 3.

FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of a substrate corresponding to one pixel ofthe array column shown in FIG. 3, in which the ISFET is shown alongsidetwo n-channel MOSFETs also included in the pixel.

FIG. 6 is a diagram similar to FIG. 5, illustrating a cross-section ofanother portion of the substrate corresponding to one pixel of the arraycolumn shown in FIG. 3, in which the ISFET is shown alongside thep-channel MOSFET of the transmission gate shown in FIG. 4.

FIG. 7 illustrates an example of a complete two-dimensional ISFET pixelarray based on the column design of FIG. 3, together with accompanyingrow and column decoder circuitry and measurement readout circuitry.

FIG. 8 generally illustrates a nucleic acid processing system comprisinga large scale chemFET array, according to one inventive embodiment ofthe present disclosure.

FIG. 9 illustrates one column of an chemFET array similar to that shownin FIG. 8, according to one inventive embodiment of the presentdisclosure.

FIG. 9A illustrates a circuit diagram for an exemplary amplifieremployed in. the array column shown in FIG. 9.

FIG. 9B is a graph of amplifier bias vs. bandwidth, according to oneinventive embodiment of the present disclosure.

FIG. 10 illustrates a top view of a chip layout design for a pixel ofthe column of an chemFET array shown in FIG. 9, according to oneinventive embodiment of the present disclosure.

FIG. 10-1 illustrates a top view of a chip layout design for a clusterof four neighboring pixels of an chemFET array shown in FIG. 9,according to another inventive embodiment of the present disclosure.

FIG. 11A shows a composite cross-sectional view along the line I-I ofthe pixel shown in FIG. 10, including additional elements on the righthalf of FIG. 10 between the lines II-II and III-III, illustrating alayer-by-layer view of the pixel fabrication according to one inventiveembodiment of the present disclosure.

FIG. 11A-1 shows a composite cross-sectional view of multipleneighboring pixels, along the line I-I of one of the pixels shown inFIG. 10-1, including additional elements of the pixel between the linesII-II, illustrating a layer-by-layer view of pixel fabrication accordingto another inventive embodiment of the present disclosure.

FIGS. 11B(1)-(3) provide the chemical structures of ten PPi receptors(compounds 1 through 10).

FIG. 11C(1) is a schematic of a synthesis protocol for compound 7 fromFIG. 11B(3).

FIG. 11C(2) is a schematic of a synthesis protocol for compound 8 fromFIG. 11B(3).

FIG. 11C(3) is a schematic of a synthesis protocol for compound 9 fromFIG. 11B(3).

FIGS. 11D(1) and (2) are schematics illustrating a variety ofchemistries that can be applied to the passivation layer in order tobind molecular recognition compounds (such as but not limited to PPireceptors).

FIG. 11E is a schematic of attachment of compound 7 from FIG. 11B(3) toa metal oxide surface.

FIGS. 12A through 12L provide top views of each of the fabricationlayers shown in FIG. 11A, according to one inventive embodiment of thepresent disclosure.

FIGS. 12-1A through 12-1L provide top views of each of the fabricationlayers shown in FIG. 11A-1, according to another inventive embodiment ofthe present disclosure.

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an chemFET sensor array similar to that shown in FIG.8, based on the column and pixel designs shown in FIGS. 9-12, accordingto one inventive embodiment of the present disclosure.

FIG. 14 illustrates a row select shift register of the array shown inFIG. 13, according to one inventive embodiment of the presentdisclosure.

FIG. 15 illustrates one of two column select shift registers of thearray shown in FIG. 13, according to one inventive embodiment of thepresent disclosure.

FIG. 16 illustrates one of two output drivers of the array shown in FIG.13, according to one inventive embodiment of the present disclosure.

FIG. 17 illustrates a block diagram of the chemFET sensor array of FIG.13 coupled to an array controller, according to one inventive embodimentof the present disclosure.

FIG. 18 illustrates an exemplary timing diagram for various signalsprovided by the array controller of FIG. 17, according to one inventiveembodiment of the present disclosure.

FIG. 18A illustrates another exemplary timing diagram for varioussignals provided by the array controller of FIG. 17, according to oneinventive embodiment of the present disclosure.

FIG. 18B shows a flow chart illustrating an exemplary method forprocessing and correction of array data acquired at high acquisitionrates, according to one inventive embodiment of the present disclosure.

FIGS. 18C and 18D illustrate exemplary pixel voltages showingpixel-to-pixel transitions in a given array output signal, according toone embodiment of the present disclosure.

FIGS. 19-20 illustrate block diagrams of alternative CMOS IC chipimplementations of chemFET sensor arrays, according to other inventiveembodiments of the present disclosure.

FIG. 20A illustrates a top view of a chip layout design for a pixel ofthe chemFET array shown in FIG. 20, according to another inventiveembodiment of the present disclosure.

FIGS. 21-23 illustrate block diagrams of additional alternative CMOS ICchip implementations of chemFET sensor arrays, according to otherinventive embodiments of the present disclosure.

FIG. 24 illustrates the pixel design of FIG. 9 implemented with ann-channel chemFET and accompanying n-channel MOSFETs, according toanother inventive embodiment of the present disclosure.

FIGS. 25-27 illustrate alternative pixel designs and associated columncircuitry for chemFET arrays according to other inventive embodiments ofthe present disclosure.

FIGS. 28A and 28B are isometric illustrations of portions of microwellarrays as employed herein, showing round wells and rectangular wells, toassist three-dimensional visualization of the array structures.

FIG. 29 is a diagrammatic depiction of a top view of one corner (i.e.,the lower left corner) of the layout of a chip showing an array ofindividual ISFET sensors on a CMOS die.

FIG. 30 is an illustration of an example of a layout for a portion of a(typically chromium) mask for a one-sensor-per-well embodiment of theabove-described sensor array, corresponding to the portion of the dieshown in FIG. 29.

FIG. 31 is a corresponding layout for a mask for a 4-sensors-per-wellembodiment.

FIG. 32 is an illustration of a second mask used to mask an area whichsurrounds the array, to build a collar or wall (or basin, using thatterm in the geological sense) of resist which surrounds the active arrayof sensors on a substrate, as shown in FIG. 33A.

FIG. 33 is an illustration of the resulting basin.

FIG. 33A is an illustration of a three-layer PCM process for making themicrowell array.

FIG. 33B is a diagrammatic cross-section of a microwell with a “bump”feature etched into the bottom.

FIG. 33B-1 is an image from a scanning electron microscope showing incross-section a portion of an array architecture as taught herein, withmicrowells formed in a layer of silicon dioxide over ISFETs.

FIG. 33B-2 is a diagrammatic illustration of a microwell incross-section, the microwell being produced as taught herein and havingsloped sides, and showing how a bead of a correspondingly appropriatediameter larger than that of the well bottom can be spaced from the wellbottom by interference with the well sidewalls.

FIG. 33B-3 is another diagrammatic illustration of such a microwell withbeads of different diameters shown, and indicating optional use ofpacking beads below the nucleic acid-carrying bead such as aDNA-carrying bead

FIGS. 34-37 diagrammatically illustrate a first example of a suitableexperiment apparatus incorporating a fluidic interface with the sensorarray, with FIG. 35 providing a cross-section through the FIG. 34apparatus along section line 35-35′ and FIG. 36 expanding part of FIG.35, in perspective, and FIG. 37 further expanding a portion of thestructure to make the fluid flow more visible.

FIG. 38 is a diagrammatic illustration of a substrate with an etchedphotoresist layer beginning the formation of an example flow cell of acertain configuration.

FIGS. 39-41 are diagrams of masks suitable for producing a firstconfiguration of flow cell consistent with FIG. 38.

FIGS. 42-54 (but not including FIGS. 42A-42L) and 57-58 are pairs ofpartly isometric, sectional views of example apparatus and enlargements,showing ways of introducing a reference electrode into, and forming, aflow cell and flow chamber, using materials such as plastic and PDMS.

FIG. 42A is an illustration of a possible cross-sectional configurationof a non-rectangular flow chamber antechamber (diffuser section) for useto promote laminar flow into a flow cell as used in the arrangementsshown herein;

FIGS. 42B-42F are diagrammatic illustrations of examples of flow cellstructures for unifying fluid flow.

FIG. 42F1 is a diagrammatic illustration of an example of a ceilingbaffle arrangement for a flow cell in which fluid is introduced at onecorner of the chip and exits at a diagonal corner, the bafflearrangement facilitating a desired fluid flow across the array.

FIGS. 42F2-42F8 comprise a set of illustrations of an exemplary flowcell member that may be manufactured by injection molding and mayincorporate baffles to facilitate fluid flow, as well as a metalizedsurface for serving as a reference electrode, including an illustrationof said member mounted to a sensor array package over a sensor array, toform a flow chamber thereover.

FIGS. 42G and 42H are diagrammatic illustrations of alternativeembodiments of flow cells in which fluid flow is introduced to themiddle of the chip assembly.

FIGS. 42I and 42J are cross-sectional illustrations of the type of flowcell embodiments shown in FIGS. 42G and 42H, mounted on a chip assembly;

FIGS. 42K and 42L are diagrammatic illustrations of flow cells in whichthe fluid is introduced at a corner of the chip assembly.

FIG. 42M is a diagrammatic illustration of fluid flow from one corner ofan array on a chip assembly to an opposite corner, in apparatus such asthat depicted in FIGS. 42K and 42L.

FIGS. 55 and 56 are schematic, cross-sectional views of two-layer glass(or plastic) arrangements for manufacturing fluidic apparatus formounting onto a chip for use as taught herein.

FIGS. 57 and 58 are schematic embodiments of a fluidic assembly.

FIGS. 59A-59C are illustrations of the pieces for two examples oftwo-piece injection molded parts for forming a flow cell.

FIG. 60 is a schematic illustration, in cross-section, for introducing astainless steel capillary tube as an electrode, into a downstream portof a flow cell such as the flow cells of FIGS. 59A-59C, or other flowcells.

FIG. 61A is a schematic illustrating the incorporation of a dNTP into asynthesized nucleic acid strand with concomitant release of inorganicpyrophosphate (PPi).

FIG. 61B is a schematic illustrating an embodiment of the invention inwhich the single stranded region of the template is not hybridized toRNA oligomers. Hydrogen ion that is released as a result of nucleotideincorporation is able to interact with and possibly be sequestered byfree bases on the single stranded region of the template. Such hydrogenions are then unable to flow to the ISFET surface and be detected. Thefree bases in the single stranded regions are proton acceptors at pHbelow 7.5.

FIG. 61C is a schematic illustrating an embodiment of the invention inwhich the single stranded region of the template is hybridized to RNAoligomers. Hydrogen ion that is released as a result of nucleotideincorporation is not able to interact with the template which ishybridized to the RNA oligomers. These hydrogen ions are therefore ableto flow to the ISFET surface and be detected.

FIG. 61D is the structure of the potassium salt of PNSE.

FIG. 61E is the structure of the sodium salt of poly(styrene sulfonicacid).

FIG. 61F is the structure of the chloride salt ofpoly(diallydimethylammonium).

FIG. 61G is the structure of the chloride salt of tetramethyl ammonium.

FIG. 61H is a schematic showing the chemistry for covalently conjugatinga primer to a bead.

FIG. 61I is a table showing the possible reactive groups that can beused in combination at positions B1, B2, P1 and P2 in order tocovalently conjugate a primer to a bead.

FIGS. 61J and K are data capture images of microwell arrays followingbead deposition. The white spots are beads. FIG. 61J is an opticalmicroscope image and FIG. 61K is an image captured using the chemFETsensor underlying the microwell array.

FIGS. 62-70 illustrate bead loading into the microfluidic arrays of theinvention.

FIG. 71 illustrates an exemplary sequencing process.

FIGS. 72A-D are graphs showing on-chip detection of nucleotideincorporation using a template of known sequence.

FIGS. 73A and B are graphs showing a trace from an ISFET device (A) anda nucleotide readout (B) from a sequencing reaction of a 23-mersynthetic oligonucleotide.

FIGS. 74A and B are graphs showing a trace from an ISFET device (A) anda nucleotide readout (B) from a sequencing reaction of a 25-mer PCRproduct.

FIG. 75A is a modeling circuit diagram for use in analyzing the factorsinfluencing ISFET gate gain;

FIG. 75B is a graph of simulated ISFET gate gain dependence onpassivation layer thickness for a first set of parameters set forth inthe specification;

FIG. 75C is a graph of simulated ISFET gate gain dependence onpassivation layer thickness for a second set of parameters set forth inthe specification;

FIG. 75D is a graph of simulated ISFET gate gain dependence onpassivation layer thickness for a third set of parameters set forth inthe specification;

FIG. 75E is a diagrammatic illustration of two microwells formed overISFETs having extended floating gate electrodes lining the walls of themicrowells;

FIG. 75F is a partially-circuit, partially diagrammatic illustration ofan example embodiment of a four-transistor pixel (sensor) employing anactive circuit design;

FIG. 75G is a diagram of a second example of a four-transistor activepixel, employing a single-MOSFET current source to avoid (or at leastminimize) introducing a disturbance at the sense node;

FIG. 75H is a diagram of a group of four pixels, each similar to that ofFIG. 75G, sharing certain components to reduce chip area requirements;

FIG. 75I is a diagram of an active pixel employing six transistors; FIG.75J is a diagram of a group of four pixels, each similar to that of FIG.75I, sharing certain components to reduce chip area requirements;

FIG. 75K is a diagrammatic illustration of an example of an array ofISFET sensors (pixels) as taught herein, sharing a commonanalog-to-digital converter (ADC) for producing digital pixel values;

FIG. 75L is a diagrammatic illustration of another example of an ISFETarray in which one ADC is provided per column (or group of columns) tospeed up digital readout;

FIGS. 75M and 75N are illustrations showing how the arrays of FIGS. 75Kand 75L may be segmented to form sub-arrays, for example to speedoperation or to treat differently different portions of the overallarray;

FIG. 75O is a partially schematic circuit, partially block diagram of asingle pixel, illustrating basically how digital output may be generatedat the individual pixel level in an array;

FIG. 75P is a diagram of a group of four pixels, each similar to that ofFIG. 75P, sharing an ADC and memory to provide per-pixel digital output;

FIG. 75Q is a diagram of row addressing circuitry and column senseamplifiers providing readout functionality from a pixel array in whichthe pixels provide digital outputs;

FIGS. 75R-75T are schematic circuit diagrams illustrating alternativesfor diode-protecting ISFETs as discussed herein;

FIG. 76A is a diagrammatic illustration of a cross-section of a firstexample of a fluid-fluid reference electrode interface in which thereference electrode is introduced downstream in the reagent path fromthe flow cell;

FIGS. 76B and 76C are diagrammatic illustrations of two alternativeexamples of ways to construct apparatus to achieve the fluid-fluidinterface of FIG. 76A;

FIG. 76D is a diagrammatic illustration of a cross-section of a secondexample of a fluid-fluid reference electrode interface in which thereference electrode is introduced upstream in the reagent path from theflow cell;

FIG. 77A is a high-level, partially block, partially circuit diagramshowing a basic passive sensor pixel in which the voltage changes on theISFET source and drain inject noise into the analyte, causing errors inthe sensed values;

FIG. 77B is a high-level partially block, partially circuit diagramshowing a basic passive sensor pixel in which the voltage changes on theISFET drain are eliminated by tying it to ground, the pixel output isobtained via a column buffer, and CDS is employed on the output of thecolumn buffer to reduce correlated noise;

FIG. 77C is a high-level partially block, partially circuit diagramshowing a two-transistor passive sensor pixel in which the voltagechanges on the ISFET drain and source are substantially eliminated, thepixel output is obtained via a buffer, and CDS is employed on the outputof the column buffer to reduce correlated noise;

FIG. 78A is an isometric, see-through, diagrammatic illustration of oneexample of a flow multiplexer for supplying fluids to a flow cell asshown herein;

FIG. 78B is a top view of the apparatus of FIG. 78A;

FIG. 78C is a diagrammatic illustration of flow through the multiplexermember of FIGS. 78A and 78B during reagent delivery mode;

FIG. 78D is a diagrammatic illustration of flow through the multiplexermember of FIGS. 78A and 78B during ship washing and reagent primingmodes

FIG. 78E is another diagrammatic illustration of wash solution flowthrough the multiplexer member;

FIG. 79 is a diagrammatic illustration of flows in the apparatus ofFIGS. 78A-78E; and

FIGS. 80A and 80B are, respectively, top and side views of analternative, “two-dimensional” fluid multiplexer.

DETAILED DESCRIPTION

Following below are more detailed descriptions of various conceptsrelated to, and embodiments of, inventive methods and apparatus relatingto large scale chemFET arrays for analyte detection and/or measurement.It should be appreciated that various concepts introduced above anddiscussed in greater detail below may be implemented in any of numerousways, as the disclosed concepts are not limited to any particular mannerof implementation. Examples of specific implementations and applicationsare provided primarily for illustrative purposes.

Various inventive embodiments according to the present disclosure aredirected at least in part to a semiconductor-based/microfluidic hybridsystem that combines the power of microelectronics with thebiocompatibility of a microfluidic system. In some examples below, themicroelectronics portion of the hybrid system is implemented in CMOStechnology for purposes of illustration. It should be appreciated,however, that the disclosure is not intended to be limiting in thisrespect, as other semiconductor-based technologies may be utilized toimplement various aspects of the microelectronics portion of the systemsdiscussed herein.

One embodiment disclosed herein is directed to a large sensor array(e.g., a two-dimensional array) of chemically-sensitive field effecttransistors (chemFETs). In related embodiments, the individual chemFETsensor elements or “pixels” of the array are configured to detectanalyte presence (or absence), analyte levels (or amounts), and/oranalyte concentration in a sample such as an unmanipulated sample, or asa result of chemical and/or biological processes (e.g., chemicalreactions, cell cultures, neural activity, nucleic acid sequencingreactions, etc.) occurring in proximity to the array. Examples ofchemFETs contemplated by various embodiments discussed in greater detailbelow include, but are not limited to, ion-sensitive field effecttransistors (ISFETs) and enzyme-sensitive field effect transistors(EnFETs). In one exemplary implementation, one or more microfluidicstructures is/are fabricated above the chemFET sensor array to providefor containment and/or confinement of a biological or chemical reactionin which an analyte of interest may be captured, produced, or consumed,as the case may be. For example, in one implementation, the microfluidicstructure(s) may be configured as one or more wells (or microwells, orreaction chambers, or reaction wells as the terms are usedinterchangeably herein) disposed above one or more sensors of the array,such that the one or more sensors over which a given well is disposeddetect and measure analyte presence, level, and/or concentration in thegiven well. Preferably, there is a 1:1 correspondence of chemFET sensorsand reaction wells.

In another exemplary implementation, the invention encompasses a systemcomprising at least one two-dimensional array of reaction chambers,wherein each reaction chamber is coupled to a chemically-sensitive fieldeffect transistor (“chemFET”) and each reaction chamber is no greaterthan 10 μm³ (i.e., 1 pL) in volume. Preferably, each reaction chamber isno greater than 0.34 pL, and more preferably no greater than 0.096 pL oreven 0.012 pL in volume. A reaction chamber can optionally be 2², 3²,4², 5², 6², 7², 8², 9², or 10² square microns in cross-sectional area atthe top. Preferably, the array has at least 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, or more reaction chambers. The reaction chambers may becapacitively coupled to the chemFETs, and preferably are capacitivelycoupled to the chemFETs. Such systems may be used for high-throughputsequencing of nucleic acids.

As used herein, an array is a planar arrangement of elements such assensors or wells. The array may be one or two dimensional. A onedimensional array is an array having one column (or row) of elements inthe first dimension and a plurality of columns (or rows) in the seconddimension. An example of a one dimensional array is a 1×5 array. A twodimensional array is an array having a plurality of columns (or rows) inboth the first and the second dimensions. The number of columns (orrows) in the first and second dimensions may or may not be the same. Anexample of a two dimensional array is a 5×10 array.

In some embodiments, such a chemFET array/microfluidics hybrid structuremay be used to analyze solution(s)/material(s) of interest containingnucleic acids. For example, such structures may be employed to sequencenucleic acids. Sequencing of nucleic acids may be performed to determinepartial or complete nucleotide sequence of a nucleic acid, to detect thepresence and in some instances nature of a mutation such as but notlimited to a single nucleotide polymorphism in a nucleic acid, toidentify source of a cell(s) or nucleic acid for example for forensicpurposes, to detect abnormal cells such as cancer cells in the bodyoptionally in the absence of detectable tumor masses, to identifypathogens in a sample such as a bodily sample for example for diagnosticand/or therapeutic purposes, to identify antibiotic resistant strains ofpathogens in order to avoid unnecessary (and ineffective) therapeuticregimens, to determine what therapeutic regimen will be most effectiveto treat a subject having a particular condition as can be determined bythe subject's genetic make-up (e.g., personalized medicine), todetermine and compare nucleic acid expression profiles of two or morestates (e.g., comparing expression profiles of diseased and normaltissue, or comparing expression profiles of untreated tissue and tissuetreated with drug, enzymes, radiation or chemical treatment), tohaplotype a sample (e.g., comparing genes or variations in genes on eachof the two alleles present in a human subject), to karyotype a sample(e.g., analyzing chromosomal make-up of a cell or a tissue such as anembryo, to detect gross chromosomal or other genomic abnormalities), andto genotype (e.g., analyzing one or more genetic loci to determine forexample carrier status and/or species-genus relationships).

The systems described herein can be utilized to sequence the nucleicacids of an entire genome, or any portion thereof. Genomes that can besequenced include mammalian genomes, and preferably human genomes. Othergenomes that can be sequenced include bacterial, viral, fungal andparasitic genomes. Such sequencing may lead to the identification ofmutations that give rise to drug resistance, or general evolutionarydrift from known species. This latter aspect is useful in determiningfor example whether a prior therapeutic (such as a vaccine) may beeffective against current infecting strains. A specific example is thedetection of new influenza strains and a determination of whether aprior year's vaccine cocktail will be effective against a new fluoutbreak.

Thus the methods of the invention may be embraced by methods fordetecting a nucleic acid in a sample. The nucleic acid may be a markerof its source such as a pathogen including but not limited to a virus,or a cancer or tumor in an individual. In the latter aspect, a samplesuch as a blood sample may be harvested from a subject and screened forthe presence of an occult cancer cell such as one that has extravasatedfrom its original tumor site. In yet another aspect, the methods may beused for forensic purposes in which samples are screened for thepresence of a known nucleic acid (e.g., from a suspect or from a lawenforcement DNA bank). In related aspects, a sample may be analyzed fornucleic acid heterogeneity in order to determine whether a sample isderived from one source (e.g., a single subject) or more than one source(e.g., a contaminated sample). The methods described herein may also beused to detect the presence of a nucleotide mutation such as but notlimited to a single nucleotide polymorphism. Such mutation analysis orscreening is typically performed in prenatal or postnatal diagnostics.The nature of the mutation can then be used to determine the mostsuitable course of therapy, in some instances. Thus the inventionintends that any of the methods provided herein can be used in one ormore diagnostic, forensic and/or therapeutic methods.

Various aspects of the invention employ a sequencing-by-synthesisapproach for sequencing nucleic acids. This approach involves thesynthesis of a new nucleic acid strand using a template nucleic acid.The template strand may be primed intermolecularly by hybridizing asequencing primer to it at one end, or intramolecularly by folding overon itself at one end. The template strand may also be primed byintroducing a break or a nick in one strand of a double-stranded nucleicacid, preferably but not exclusively near an end, as described ingreater detail herein. In these embodiments, known nucleotides areincorporated into the “primer” based on complementarity with thetemplate. The method requires that nucleotides be contacted with theprimer (and thus template) (in the presence of polymerase and any otherfactors required for incorporation) in a selective manner.

In many embodiments, each nucleotide type is individually contacted withthe primer and/or template. In other embodiments, combinations of two orthree types of nucleotides may be contacted with the primer and/ortemplate simultaneously. Since the identity of the nucleotides incontact with the primer and/or template at any given time is known, theidentity of the incorporated nucleotides (if incorporated) is alsoknown. And based on the necessary complementarity with the template, thesequence of the template can also be deduced. Using different types ofnucleotides separately (e.g., using dATP, dCTP, dGTP or dTTP separatelyfrom each other), a high resolution sequence can be obtained. Usingcombinations (or mixtures) of nucleotides (e.g., using dATP, dCTP anddGTP together and separately from dTTP), a lower resolution sequence canbe obtained that is nevertheless valuable for certain applications(e.g., ordering and aligning various higher resolution sequences). Withregards to the latter embodiment, it will be understood that theinvention contemplates using mixtures of any three nucleotides, and insome cases any two nucleotides, and not just the specific combinationsrecited above.

The nucleotides (or nucleotide triphosphates or deoxyribonucleotides ordNTPs, as they are referred to herein interchangeably) need not be andtypically are not extrinsically labeled. Thus, naturally occurringnucleotides (i.e., nucleotides identical to those that exist in vivonaturally) or their synthetic counterparts are suitable for use in themethods of the invention. Such nucleotides may be referred to herein asbeing “unlabeled”.

Preferably, the nucleotides are delivered at substantially the same timeto each template. Polymerase(s) are preferably already present, althoughthey also may be introduced along with the nucleotides. The polymerasesmay be immobilized or may be free flowing. Once the nucleotides areincorporated (if complementarity exists) and any associated signal isdetected, an enzyme, such as apyrase, is typically delivered to degradeany unused nucleotides, followed by a washing step to removesubstantially all of the enzyme as well as any other remaining andundesirable components. The reaction may occur in a reaction chamber insome embodiments, while in others it may occur in the absence ofreaction chambers. In these latter embodiments, the sensor surface maybe continuous without any physical divider between sensors.

In important embodiments, the sequencing reaction is performedsimultaneously on a plurality of identical templates in a reactionchamber, and optionally in a plurality of reaction chambers. Sequencinga different template in each reaction chamber allows a greater amount ofsequence data to be obtained in any given run. Thus, using as manyreaction chambers (and sensors) as possible in a given run alsomaximizes the amount of sequence data that can be obtained in any givenrun. In important embodiments, the templates in a reaction well areimmobilized (e.g., covalently or non-covalently) onto and/or in a bead,referred to herein as a capture bead, or onto a solid support such asthe chemFET surface.

It is to be understood that in this and other embodiments and aspects ofthe invention, a plurality may represent a subset of elements ratherthan the entirety of all elements. As an example, in the aboveembodiment, the plurality of templates in the reaction chamber that aresequenced may represent a subset or all of the templates in the reactionchamber. Thus this particular embodiment requires that at least twotemplates be sequenced, and it does not require that all the templatespresent in the reaction chamber be sequenced.

As described extensively herein, in some embodiments, nucleotideincorporation is detected through byproducts of the incorporation or bychanges in charge to the newly synthesized nucleic acid, especiallywhere it is immobilized on a chemFET surface, rather than by detectingthe incorporated nucleotide itself. More specifically, some embodimentsexploit the release of inorganic pyrophosphate (PPi), inorganicphosphate (Pi), and hydrogen ions (all of which are consideredsequencing reaction byproducts) that occurs following incorporation of anucleotide into a nucleic acid (such as a primer, for example). In someembodiments of the invention, the method detects the released hydrogenions as an indication of nucleotide incorporation. The chemFETs (andchemFET arrays) described herein are suited to the detection of theseions as well as other sequencing reaction byproducts. It is to beunderstood that the aspects and embodiments described herein related tochemFETs equally contemplate and embrace ISFETs unless otherwise stated.

The invention includes methods for improving detection of the hydrogenions by the chemFET. These methods include generating and/or detectingmore hydrogen ions in a given sequencing reaction. This can be done byincreasing the number of templates per reaction chamber, increasing thenumber of templates attached to each capture bead, increasing the numberof templates being sequenced per reaction chamber, increasing the numberof templates bound to the sensor surface, increasing the stability ofthe primer/template hybrid, increasing the processivity of thepolymerase, and/or combining nucleotide incorporation with nucleotideexcision (e.g., performing the sequencing-by-synthesis reaction in thecontext of a nick translation reaction), among other things. Anotheralternative or additional approach is to increase the number of releasedhydrogen ions that are actually detected by the chemFET. This can bedone by preventing the released hydrogen ions from interacting withother components in the reaction well including any components withbuffering potential. These embodiments include using bufferinginhibitors (as described more fully herein) to saturate components thatmight otherwise sequester released hydrogen ions. Buffering inhibitorsmay be short RNA oligomers that bind to single stranded regions of thetemplates, or chemical compounds that interact with the materialscomprised in the reaction chambers and/or chemFETs themselves.

Some aspects and embodiments presented herein involve dense chemFETarrays and reaction chamber arrays. It will be apparent that as arraysbecome more dense, area and/or volume of individual elements (e.g.,sensor surfaces and reaction chambers) will typically become smaller inorder to accommodate a greater number of sensors or reaction chamberswithout a concomitant (or significant) increase in total array area.However, it has been determined in accordance with an aspect of theinvention that as volume of a reaction chamber decreases, the signal tonoise ratio can actually increase due to an increased nucleic acidconcentration. For example, it has been determined that a roughly 2.3fold decrease in reaction chamber volume can yield about a 1.5 foldincrease in signal to noise ratio. This increase can occur even if thetotal number of nucleic acids being sequenced is reduced. Thus, in someinstances rather than losing signal by moving to more dense arrays, theinvention contemplates a greater signal due to an increasedconcentration of nucleic acids in the smaller volume reaction chambers.

The invention also contemplates sequencing-by-synthesis methods thatdetect nucleotide incorporation events based on changes in charge at thechemFET surface due to the a change in charge of a moiety attached tothe surface, such as a nucleic acid or a nucleic acid complex (e.g., atemplate/primer hybrid). Such methods include those that use or extendnucleic acids that are immobilized (e.g., covalently) to the surface ofa chemFET. Nucleotide incorporation into a nucleic acid that is bound toa chemFET surface typically results in an increase in the negativecharge of the bound nucleic acid or the complex in which it is present(e.g., a template/primer hybrid). In some instances, the primer will bebound to the chemFET surface while in other instances the template willbe bound to the chemFET surface. In such instances, a plurality ofidentical, typically physically separate, nucleic acids are immobilizedto individual chemFET surfaces and sequencing-by-synthesis reactions areperformed on the plurality simultaneously and synchronously. In someembodiments, the nucleic acids are not concatemers and rather each willinclude only a single copy of the nucleic acid to be sequenced.

It will be understood that the sequencing methods provided herein can beused to sequence a genome or part thereof. As an example, such a methodmay include delivering fragmented nucleic acids from the genome or partthereof to a system for high-throughput sequencing comprising at leastone array of reaction chambers, wherein each reaction chamber is coupledto a chemFET, and detecting a sequencing reaction in a reaction chambervia a signal from the chemFET coupled with the reaction chamber.Alternatively, the method may include delivering fragmented nucleicacids from the genome or part thereof to a sequencing apparatuscomprising an array of reaction chambers, wherein each of the reactionchambers is disposed in a sensing relationship with an individualassociated chemFET, and detecting a sequencing reaction a reactionchambers via a signal from its associated chemFET. Typically, all fournucleotides are flowed into the same reaction chamber, eitherindividually (or separately) or as some mixture of less than all fournucleotides, in an ordered and known manner.

The methods provided herein may allow for at least 10³, preferably atleast 10⁴, more preferably at least 10⁵, and even more preferably atleast 10⁶ bases to be determined (or sequenced) per hour. In even morepreferred embodiments, at least 10⁷ bases, at least 10⁸ bases, at least10⁹ bases, or at least 10¹⁰ bases are sequenced per hour using themethods and arrays discussed herein. Thus, the methods may be used tosequence an entire human genome within about 24 hours, more preferablywithin about 20 hours, even more preferably within about 15 hours, evenmore preferably within about 10 hours, even more preferably within about5 hours, and most preferably within about 1 hour.

It should be appreciated, however, that while some illustrative examplesof the concepts disclosed herein focus on nucleic acid sequencing, theinvention contemplates a broader application of these methods and is notintended to be limited to these examples.

FIG. 8 generally illustrates a nucleic acid processing system 1000comprising a large scale chemFET array, according to one inventiveembodiment of the present disclosure. An example of a nucleic acidprocessing system is a nucleic acid sequencing system. In the discussionthat follows, the chemFET sensors of the array are described forpurposes of illustration as ISFETs configured for sensitivity to staticand/or dynamic ion concentration, including but not limited to hydrogenion concentration. However, it should be appreciated that the presentdisclosure is not limited in this respect, and that in any of theembodiments discussed herein in which ISFETs are employed as anillustrative example, other types of chemFETs may be similarly employedin alternative embodiments, as discussed in further detail below.Similarly it should be appreciated that various aspects and embodimentsof the invention may employ ISFETs as sensors yet detect one or moreionic species that are not hydrogen ions.

The system 1000 includes a semiconductor/microfluidics hybrid structure300 comprising an ISFET sensor array 100 and a microfluidics flow cell200. In one aspect, the flow cell 200 may comprise a number of wells(not shown in FIG. 8) disposed above corresponding sensors of the ISFETarray 100. In another aspect, the flow cell 200 is configured tofacilitate the sequencing of one or more identical template nucleicacids disposed in the flow cell via the controlled and orderedintroduction to the flow cell of a number of sequencing reagents 272(e.g., dATP, dCTP, dGTP, dTTP (generically referred to herein as dNTP),divalent cations such as but not limited to Mg²⁺, wash solutions, andthe like).

As illustrated in FIG. 8, the introduction of the sequencing reagents tothe flow cell 200 may be accomplished via one or more valves 270 and oneor more pumps 274 that are controlled by a computer 260. A number oftechniques may be used to admit (i.e., introduce) the various processingmaterials (i.e., solutions, samples, reaction reagents, wash solutions,and the like) into the wells of such a flow cell. As illustrated in FIG.8, reagents including dNTP may be admitted to the flow cell (e.g., viathe computer controlled valve 270 and pumps 274) from which they diffuseinto the wells, or reagents may be added to the flow cell by other meanssuch as an ink jet. In yet another example, the flow cell 200 may notcontain any wells, and diffusion properties of the reagents may beexploited to limit cross-talk between respective sensors of the ISFETarray 100, or nucleic acids may be immobilized on the surfaces ofsensors of the ISFET array 100.

The flow cell 200 in the system of FIG. 8 may be configured in a varietyof manners to provide one or more analytes (or one or more reactionsolutions) in proximity to the ISFET array 100. For example, a templatenucleic acid may be directly attached or applied in suitable proximityto one or more pixels of the sensor array 100, or in or on a supportmaterial (e.g., one or more “beads”) located above the sensor array butwithin the reaction chambers, or on the sensor surface itself.Processing reagents (e.g., enzymes such as polymerases) can also beplaced on the sensors directly, or on one or more solid supports (e.g.,they may be bound to the capture beads or to other beads) in proximityto the sensors, or they may be in solution and free-flowing. It is to beunderstood that the device may be used without wells or beads.

In the system 1000 of FIG. 8, according to one embodiment the ISFETsensor array 100 monitors ionic species, and in particular, changes inthe levels/amounts and/or concentration of ionic species, includinghydrogen ions. In important embodiments, the species are those thatresult from a nucleic acid synthesis or sequencing reaction.

Various embodiments of the present invention may relate tomonitoring/measurement techniques that involve the static and/or dynamicresponses of an ISFET. It is to be understood that although theparticular example of a nucleic acid synthesis or sequencing reaction isprovided to illustrate the transient or dynamic response of chemFET suchas an ISFET, the transient or dynamic response of a chemFET such as anISFET as discussed below may be exploited for monitoring/sensing othertypes of chemical and/or biological activity beyond the specific exampleof a nucleic acid synthesis or sequencing reaction.

As noted above, the ISFET may be employed to measure steady state pHvalues, since in some embodiments pH change is proportional to thenumber of nucleotides incorporated into the newly synthesized nucleicacid strand. In other embodiments discussed in greater detail below, theFET sensor array may be particularly configured for sensitivity to otheranalytes that may provide relevant information about the chemicalreactions of interest. An example of such a modification orconfiguration is the use of analyte-specific receptors to bind theanalytes of interest, as discussed in greater detail herein.

Via an array controller 250 (also under operation of the computer 260),the ISFET array may be controlled so as to acquire data (e.g., outputsignals of respective ISFETs of the array) relating to analyte detectionand/or measurements, and collected data may be processed by the computer260 to yield meaningful information associated with the processing(including sequencing) of the template nucleic acid.

With respect to the ISFET array 100 of the system 1000 shown in FIG. 8,in one embodiment the array 100 is implemented as an integrated circuitdesigned and fabricated using standard CMOS processes (e.g., 0.35micrometer process, 0.18 micrometer process), comprising all the sensorsand electronics needed to monitor/measure one or more analytes and/orreactions. With reference again to FIG. 1, one or more referenceelectrodes 76 to be employed in connection with the ISFET array 100 maybe placed in the flow cell 200 (e.g., disposed in “unused” wells of theflow cell) or otherwise exposed to a reference (e.g., one or more of thesequencing reagents 172) to establish a base line against which changesin analyte concentration proximate to respective ISFETs of the array 100are compared. The reference electrode(s) 76 may be electrically coupledto the array 100, the array controller 250 or directly to the computer260 to facilitate analyte measurements based on voltage signals obtainedfrom the array 100; in some implementations, the reference electrode(s)may be coupled to an electric ground or other predetermined potential,or the reference electrode voltage may be measured with respect toground, to establish an electric reference for ISFET output signalmeasurements, as discussed further below.

The ISFET array 100 is not limited to any particular size, as one- ortwo-dimensional arrays, including but not limited to as few as two to256 pixels (e.g., 16 by 16 pixels in a two-dimensional implementation)or as many as 54 mega-pixels (e.g., 7400 by 7400 pixels in atwo-dimensional implementation) or even greater may be fabricated andemployed for various chemical/biological analysis purposes pursuant tothe concepts disclosed herein. In one embodiment of the exemplary systemshown in FIG. 8, the individual ISFET sensors of the array may beconfigured for sensitivity to hydrogen ions; however, it should also beappreciated that the present disclosure is not limited in this respect,as individual sensors of an ISFET sensor array may be particularlyconfigured for sensitivity to other types of ion concentrations for avariety of applications (materials sensitive to other ions such assodium, silver, iron, bromine, iodine, calcium, and nitrate, forexample, are known).

More generally, a chemFET array according to various embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes and/or one or more binding events, and in other embodimentsdifferent chemFETs of a given array may be configured for sensitivity todifferent analytes. For example, in one embodiment, one or more sensors(pixels) of the array may include a first type of chemFET configured tobe sensitive to a first analyte, and one or more other sensors of thearray may include a second type of chemFET configured to be sensitive toa second analyte different from the first analyte. In one exemplaryimplementation, both a first and a second analyte may indicate aparticular reaction such as for example nucleotide incorporation in asequencing-by-synthesis method. Of course, it should be appreciated thatmore than two different types of chemFETs may be employed in any givenarray to detect, and/or measure different types of analytes and/or otherreactions. In general, it should be appreciated in any of theembodiments of sensor arrays discussed herein that a given sensor arraymay be “homogeneous” and include chemFETs of substantially similar oridentical types to detect and/or measure a same type of analyte (e.g.,hydrogen ions), or a sensor array may be “heterogeneous” and includechemFETs of different types to detect and/or measure different analytes.For simplicity of discussion, again the example of an ISFET is discussedbelow in various embodiments of sensor arrays, but the presentdisclosure is not limited in this respect, and several other options foranalyte sensitivity are discussed in further detail below (e.g., inconnection with FIG. 11A).

The chemFET arrays configured for sensitivity to any one or more of avariety of analytes may be disposed in electronic chips, and each chipmay be configured to perform one or more different biological reactions.The electronic chips can be connected to the portions of theabove-described system which read the array output by means of pinscoded in a manner such that the pins convey information to the system asto characteristics of the array and/or what kind of biologicalreaction(s) is (are) to be performed on the particular chip.

In one embodiment, the invention encompasses an electronic chipconfigured for conducting biological reactions thereon, comprising oneor more pins for delivering information to a circuitry identifying acharacteristic of the chip and/or a type of reaction to be performed onthe chip. Such reactions or applications may include, but are notlimited to, nucleotide polymorphism detection, short tandem repeatdetection, or general sequencing.

In another embodiment, the invention encompasses a system adapted toperform more than one biological reaction on a chip the systemcomprising a chip receiving module adapted for receiving the chip, and areceiver for detecting information from the electronic chip, wherein theinformation determines a biological reaction to be performed on thechip. Typically, the system further comprises one or more reagents toperform the selected biological reaction.

In another embodiment, the invention encompasses an apparatus forsequencing a polymer template comprising at least one integrated circuitthat is configured to relay information about spatial location of areaction chamber, the type of monomer added to the spatial location, andthe time required to complete reaction of a reagent comprising aplurality of the monomers with an elongating polymer.

In exemplary implementations based on 0.35 micrometer CMOS processingtechniques (or CMOS processing techniques capable of smaller featuresizes), each pixel of the ISFET array 100 may include an ISFET andaccompanying enable/select components, and may occupy an area on asurface of the array of approximately ten micrometers by ten micrometers(i.e., 100 micrometers²) or less; stated differently, arrays having apitch (center of pixel-to-center of pixel spacing) on the order of 10micrometers or less may be realized. An array pitch on the order of 10micrometers or less using a 0.35 micrometer CMOS processing techniqueconstitutes a significant improvement in terms of size reduction withrespect to prior attempts to fabricate ISFET arrays, which resulted inpixel sizes on the order of at least 12 micrometers or greater.

More specifically, in some embodiments discussed further below based onthe inventive concepts disclosed herein, an array pitch of approximatelynine (9) micrometers allows an ISFET array including over 256,000 pixels(e.g., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (e.g., a 2048 by 2048 array) to be fabricatedon a 21 millimeter by 21 millimeter die. In other examples, an arraypitch of approximately 5 micrometers allows an ISFET array includingapproximately 1.55 Mega-pixels (e.g., a 1348 by 1152 array) andassociated electronics to be fabricated on a 9 millimeter by 9millimeter die, and an ISFET sensor array including over 14 Mega-pixelsand associated electronics on a 22 millimeter by 20 millimeter die. Inyet other implementations, using a CMOS fabrication process in whichfeature sizes of less than 0.35 micrometers are possible (e.g., 0.18micrometer CMOS processing techniques), ISFET sensor arrays with a pitchsignificantly below 5 micrometers may be fabricated (e.g., array pitchof 2.6 micrometers or pixel area of less than 8 or 9 micrometers²),providing for significantly dense ISFET arrays.

As will be understood by those of skill in the art, the ability tominiaturize sequencing reactions reduces the time, cost and laborinvolved in sequencing of large genomes (such as the human genome). Ofcourse, it should be appreciated that pixel sizes greater than 10micrometers (e.g., on the order of approximately 20, 50, 100 micrometersor greater) may be implemented in various embodiments of chemFET arraysaccording to the present disclosure also.

In other aspects of the system shown in FIG. 8, one or more arraycontrollers 250 may be employed to operate the ISFET array 100 (e.g.,selecting/enabling respective pixels of the array to obtain outputsignals representing analyte measurements). In various implementations,one or more components constituting one or more array controllers may beimplemented together with pixel elements of the arrays themselves, onthe same integrated circuit (IC) chip as the array but in a differentportion of the IC chip, or off-chip. In connection with array control,analog-to-digital conversion of ISFET output signals may be performed bycircuitry implemented on the same integrated circuit chip as the ISFETarray, but located outside of the sensor array region (locating theanalog to digital conversion circuitry outside of the sensor arrayregion allows for smaller pitch and hence a larger number of sensors, aswell as reduced noise). In various exemplary implementations discussedfurther below, analog-to-digital conversion can be 4-bit, 8-bit, 12-bit,16-bit or other bit resolutions depending on the signal dynamic rangerequired.

In general, data may be removed from the array in serial or parallel orsome combination thereof. On-chip controllers (or sense amplifiers) cancontrol the entire chip or some portion of the chip. Thus, the chipcontrollers or signal amplifiers may be replicated as necessaryaccording to the demands of the application. The array may, but need notbe, uniform. For instance, if signal processing or some other constraintrequires instead of one large array multiple smaller arrays, each withits own sense amplifiers or controller logic, that is quite feasible.

Having provided a general overview of the role of a chemFET (e.g.,ISFET) array 100 in an exemplary system 1000 for measuring one or moreanalytes, following below are more detailed descriptions of exemplarychemFET arrays according to various inventive embodiments of the presentdisclosure that may be employed in a variety of applications. Again, forpurposes of illustration, chemFET arrays according to the presentdisclosure are discussed below using the particular example of an ISFETarray, but other types of chemFETs may be employed in alternativeembodiments. Also, again, for purposes of illustration, chemFET arraysare discussed in the context of nucleic acid sequencing applications,however, the invention is not so limited and rather contemplates avariety of applications for the chemFET arrays described herein.

As noted above, various inventive embodiments disclosed hereinspecifically improve upon the ISFET array design of Milgrew et al.discussed above in connection with FIGS. 1-7, as well as other priorISFET array designs, so as to significantly reduce pixel size and arraypitch, and thereby increase the number of pixels of an ISFET array for agiven semiconductor die size (i.e., increase pixel density). In someimplementations, an increase in pixel density is accomplished while atthe same time increasing the signal-to-noise ratio (SNR) of outputsignals corresponding to respective measurements relating to one or moreanalytes and the speed with which such output signals may be read fromthe array. In particular, by relaxing requirements for ISFET linearityand focusing on a more limited signal output/measurement range (e.g.,signal outputs corresponding to a pH range of from approximately 7 to 9or smaller rather than 1 to 14, as well as output signals that may notnecessarily relate significantly to pH changes in sample), individualpixel complexity and size may be significantly reduced, therebyfacilitating the realization of very large scale dense ISFET arrays.

To this end, FIG. 9 illustrates one column 102 _(j) of an ISFET array100, according to one inventive embodiment of the present disclosure, inwhich ISFET pixel design is appreciably simplified to facilitate smallpixel size. The column 102 _(j) includes n pixels, the first and last ofwhich are shown in FIG. 9 as the pixels 105 ₁ and 105 _(n). As discussedfurther below in connection with FIG. 13, a complete two-dimensionalISFET array 100 based on the column design shown in FIG. 9 includes msuch columns 102 _(j) (j=1, 2, 3, . . . m) with successive columns ofpixels generally arranged side by side. Of course, the ISFETs may bearrayed in other than a row-column grid, such as in a honeycomb pattern.

In one aspect of the embodiment shown in FIG. 9, each pixel 105 ₁,through 105 _(n) of the column 102 _(j) includes only three components,namely, an ISFET 150 (also labeled as Q1) and two MOSFET switches Q2 andQ3. The MOSFET switches Q2 and Q3 are both responsive to one of n rowselect signals ( RowSel₁ through RowSel_(n) , logic low active) so as toenable or select a given pixel of the column 102 _(j). Using pixel 105 ₁as an example that applies to all pixels of the column, the transistorswitch Q3 couples a controllable current source 106 _(j) via the line112 ₁ to the source of the ISFET 150 upon receipt of the correspondingrow select signal via the line 118 ₁. The transistor switch Q2 couplesthe source of the ISFET 150 to column bias/readout circuitry 110 _(j)via the line 114 ₁ upon receipt of the corresponding row select signal.The drain of the ISFET 150 is directly coupled via the line 116 ₁ to thebias/readout circuitry 110 _(j). Thus, only four signal lines per pixel,namely the lines 112 ₁, 114 ₁, 116 ₁ and 118 ₁, are required to operatethe three components of the pixel 105 ₁. In an array of m columns, agiven row select signal is applied simultaneously to one pixel of eachcolumn (e.g., at same positions in respective columns).

As illustrated in FIG. 9, the design for the column 102 _(j) accordingto one embodiment is based on general principles similar to thosediscussed above in connection with the column design of Milgrew et al.shown FIG. 3. In particular, the ISFET of each pixel, when enabled, isconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel according to Eq. (3) above. To this end, the column 102_(j) includes a controllable current source 106 _(j), coupled to ananalog circuitry positive supply voltage VDDA and responsive to a biasvoltage VB1, that is shared by all pixels of the column to provide aconstant drain current I_(Dj) to the ISFET of an enabled pixel. In oneaspect, the current source 106 _(j) is implemented as a current mirrorincluding two long-channel length and high output impedance MOSFETs. Thecolumn also includes bias/readout circuitry 110 _(j) that is also sharedby all pixels of the column to provide a constant drain-to-sourcevoltage to the ISFET of an enabled pixel. The bias/readout circuitry 110_(j) is based on a Kelvin Bridge configuration and includes twooperational amplifiers 107A (A1) and 107B (A2) configured as bufferamplifiers and coupled to analog circuitry positive supply voltage VDDAand the analog supply voltage ground VSSA. The bias/readout circuitryalso includes a controllable current sink 108 _(j) (similar to thecurrent source 106 _(j)) coupled to the analog ground VSSA andresponsive to a bias voltage VB2, and a diode-connected MOSFET Q6. Thebias voltages VB1 and VB2 are set/controlled in tandem to provide acomplimentary source and sink current. The voltage developed across thediode-connected MOSFET Q6 as a result of the current drawn by thecurrent sink 108 _(j) is forced by the operational amplifiers to appearacross the drain and source of the ISFET of an enabled pixel as aconstant drain-source voltage V_(DSj).

By employing the diode-connected MOSFET Q6 in the bias/readout circuitry110 _(j) of FIG. 9, rather than the resistor R_(SDj) as shown in thedesign of Milgrew et al. illustrated in FIG. 3, a significant advantageis provided in a CMOS fabrication process; specifically, matchingresistors can be fabricated with error tolerances generally on the orderof ±20%, whereas MOSFET matching in a CMOS fabrication process is on theorder of ±1% or better. The degree to which the component responsiblefor providing a constant ISFET drain-to-source voltage V_(DSj) can bematched from column to column significantly affects measurement accuracy(e.g., offset) from column to column. Thus, employing the MOSFET Q6rather than a resistor appreciably mitigates measurement offsets fromcolumn-to-column. Furthermore, whereas the thermal drift characteristicsof a resistor and an ISFET may be appreciably different, the thermaldrift characteristics of a MOSFET and ISFET are substantially similar,if not virtually identical; hence, any thermal drift in MOSFET Q6virtually cancels any thermal drift from ISFET Q1, resulting in greatermeasurement stability with changes in array temperature.

In FIG. 9, the column bias/readout circuitry 110 j also includessample/hold and buffer circuitry to provide an output signal V_(COLj)from the column. In particular, after one of the pixels 105 ₁ through105 _(n) is enabled or selected via the transistors Q2 and Q3 in eachpixel, the output of the amplifier 107A (A1), i.e., a buffered V_(Sj),is stored on a column sample and hold capacitor C_(sh) via operation ofa switch (e.g., a transmission gate) responsive to a column sample andhold signal COL SH. Examples of suitable capacitances for the sample andhold capacitor include, but are not limited to, a range of fromapproximately 500 fF to 2 pF. The sampled voltage is buffered via acolumn output buffer amplifier 111 j (BUF) and provided as the columnoutput signal V_(COLj). As also shown in FIG. 9, a reference voltageVREF may be applied to the buffer amplifier 111 j, via a switchresponsive to a control signal CAL, to facilitate characterization ofcolumn-to-column non-uniformities due to the buffer amplifier 111 j andthus allow post-read data correction.

FIG. 9A illustrates an exemplary circuit diagram for one of theamplifiers 107A of the bias/readout circuitry 110 j (the amplifier 107Bis implemented identically), and FIG. 9B is a graph of amplifier biasvs. bandwidth for the amplifiers 107A and 107B. As shown in FIG. 9A, theamplifier 107A employs an arrangement of multiple current mirrors basedon nine MOSFETs (M1 through M9) and is configured as a unity gainbuffer, in which the amplifier's inputs and outputs are labeled forgenerality as IN+ and VOUT, respectively. The bias voltage VB4(representing a corresponding bias current) controls the transimpedanceof the amplifier and serves as a bandwidth control (i.e., increasedbandwidth with increased current). With reference again to FIG. 9, dueto the sample and hold capacitor C_(sh), the output of the amplifier107A essentially drives a filter when the sample and hold switch isclosed. Accordingly, to achieve appreciably high data rates, the biasvoltage VB4 may be adjusted to provide higher bias currents andincreased amplifier bandwidth. From FIG. 9B, it may be observed that insome exemplary implementations, amplifier bandwidths of at least 40 MHzand significantly greater may be realized. In some implementations,amplifier bandwidths as high as 100 MHz may be appropriate to facilitatehigh data acquisition rates and relatively lower pixel sample or “dwell”times (e.g., on the order of 10 to 20 microseconds).

In another aspect of the embodiment shown in FIG. 9, unlike the pixeldesign of Milgrew et al. shown in FIG. 3, the pixels 105 ₁ through 105_(n) do not include any transmission gates or other devices that requireboth n-channel and p-channel FET components; in particular, the pixels105 ₁ through 105 _(n) of this embodiment include only FET devices of asame type (i.e., only n-channel or only p-channel). For purposes ofillustration, the pixels 105 ₁ and 105 _(n) illustrated in FIG. 9 areshown as comprising only p-channel components, i.e., two p-channelMOSFETs Q2 and Q3 and a p-channel ISFET 150. By not employing atransmission gate to couple the source of the ISFET to the bias/readoutcircuitry 110 _(j), some dynamic range for the ISFET output signal(i.e., the ISFET source voltage V_(S)) may be sacrificed. However, bypotentially foregoing some output signal dynamic range (and therebypotentially limiting measurement range for a given static and/or dynamicchemical property, such as pH), the requirement of different type FETdevices (both n-channel and p-channel) in each pixel may be eliminatedand the pixel component count reduced. As discussed further below inconnection with FIGS. 10-12, this significantly facilitates pixel sizereduction. Thus, in one aspect, there is a beneficial tradeoff betweenreduced dynamic range and smaller pixel size.

In yet another aspect of the embodiment shown in FIG. 9, unlike thepixel design of Milgrew et al., the ISFET 150 of each pixel 105 ₁through 105 _(n) does not have its body connection tied to its source(i.e., there is no electrical conductor coupling the body connection andsource of the ISFET such that they are forced to be at the same electricpotential during operation). Rather, the body connections of all ISFETsof the array are tied to each other and to a body bias voltage V_(BODY).While not shown explicitly in FIG. 9, the body connections for theMOSFETs Q2 and Q3 likewise are not tied to their respective sources, butrather to the body bias voltage V_(BODY). In one exemplaryimplementation based on pixels having all p-channel components, the bodybias voltage V_(BODY) is coupled to the highest voltage potentialavailable to the array (e.g., VDDA), as discussed further below inconnection with FIG. 17.

By not tying the body connection of each ISFET to its source, thepossibility of some non-zero source-to-body voltage V_(SB) may give riseto the “body effect,” as discussed above in connection with FIG. 1,which affects the threshold voltage V_(TH) of the ISFET according to anonlinear relationship (and thus, according to Eqs. (3), (4) and (5) mayaffect detection and/or measurement of analyte activity giving rise tosurface potential changes at the analyte/passivation layer interface).However, by focusing on a reduced ISFET output signal dynamic range, anybody effect that may arise in the ISFET from a non-zero source-to-bodyvoltage may be relatively minimal. Thus, any measurement nonlinearitythat may result over the reduced dynamic range may be ignored asinsignificant or taken into consideration and compensated (e.g., viaarray calibration and data processing techniques, as discussed furtherbelow in connection with FIG. 17). By not tying each ISFET source to itsbody connection, all of the FETs constituting the pixel may share acommon body connection, thereby further facilitating pixel sizereduction, as discussed further below in connection with FIGS. 10-12.Accordingly, in another aspect, there is a beneficial tradeoff betweenreduced linearity and smaller pixel size.

FIG. 10 illustrates a top view of a chip layout design for the pixel 105₁ shown in FIG. 9, according to one inventive embodiment of the presentdisclosure. FIG. 11A shows a composite cross-sectional view along theline I-I of the pixel shown in FIG. 10, including additional elements onthe right half of FIG. 10 between the lines II-II and III-III,illustrating a layer-by-layer view of the pixel fabrication, and FIGS.12A through 12L provide top views of each of the fabrication layersshown in FIG. 11A (the respective images of FIGS. 12A through 12L aresuperimposed one on top of another to create the pixel chip layoutdesign shown in FIG. 10). In one exemplary implementation, the pixeldesign illustrated in FIGS. 10-12 may be realized using a standard4-metal, 2-poly, 0.35 micrometer CMOS process to provide a geometricallysquare pixel having a dimension “e” as shown in FIG. 10 of approximately9 micrometers, and a dimension “f” corresponding to the ISFET sensitivearea of approximately 7 micrometers.

In the top view of FIG. 10, the ISFET 150 (labeled as Q1 in FIG. 10)generally occupies the right center portion of the pixel illustration,and the respective locations of the gate, source and drain of the ISFETare indicated as Q1 _(G), Q1 _(S) and Q1 _(D). The MOSFETs Q2 and Q3generally occupy the left center portion of the pixel illustration; thegate and source of the MOSFET Q2 are indicated as Q2 _(G) and Q2 _(S),and the gate and source of the MOSFET Q3 are indicated as Q3 _(G) and Q3_(S). In one aspect of the layout shown in FIG. 10, the MOSFETs Q2 andQ3 share a drain, indicated as Q2/3 _(D). In another aspect, it may beobserved generally from the top view of FIG. 10 that the ISFET is formedsuch that its channel lies along a first axis of the pixel (e.g.,parallel to the line I-I), while the MOSFETs Q2 and Q3 are formed suchthat their channels lie along a second axis perpendicular to the firstaxis. FIG. 10 also shows the four lines required to operate the pixel,namely, the line 112 ₁ coupled to the source of Q3, the line 114 ₁coupled to the source of Q2, the line 116 ₁ coupled to the drain of theISFET; and the row select line 118 ₁ coupled to the gates of Q2 and Q3.With reference to FIG. 9, it may be appreciated that all pixels in agiven column share the lines 112, 114 and 116 (e.g., running verticallyacross the pixel in FIG. 10), and that all pixels in a given row sharethe line 118 (e.g., running horizontally across the pixel in FIG. 10);thus, based on the pixel design of FIG. 9 and the layout shown in FIG.10, only four metal lines need to traverse each pixel.

With reference now to the cross-sectional view of FIG. 11A, highly dopedp-type regions 156 and 158 (lying along the line I-I in FIG. 10) inn-well 154 constitute the source (S) and drain (D) of the ISFET, betweenwhich lies a region 160 of the n-well in which the ISFETs p-channel isformed below the ISFETs polysilicon gate 164 and a gate oxide 165.According to one aspect of the inventive embodiment shown in FIGS. 10and 11, all of the FET components of the pixel 105 ₁ are fabricated asp-channel FETs in the single n-type well 154 formed in a p-typesemiconductor substrate 152. This is possible because, unlike the designof Milgrew et al., 1) there is no requirement for a transmission gate inthe pixel; and 2) the ISFETs source is not tied to the n-well's bodyconnection. More specifically, highly doped n-type regions 162 provide abody connection (B) to the n-well 154 and, as shown in FIG. 10, the bodyconnection B is coupled to a metal conductor 322 around the perimeter ofthe pixel 105 ₁. However, the body connection is not directlyelectrically coupled to the source region 156 of the ISFET (i.e., thereis no electrical conductor coupling the body connection and source suchthat they are forced to be at the same electric potential duringoperation), nor is the body connection directly electrically coupled tothe gate, source or drain of any component in the pixel. Thus, the otherp-channel FET components of the pixel, namely Q2 and Q3, may befabricated in the same n-well 154.

In the composite cross-sectional view of FIG. 11A, a highly doped p-typeregion 159 is also visible (lying along the line I-I in FIG. 10),corresponding to the shared drain (D) of the MOSFETs Q2 and Q3. Forpurposes of illustration, a polysilicon gate 166 of the MOSFET Q3 alsois visible in FIG. 11A, although this gate does not lie along the lineI-I in FIG. 10, but rather “behind the plane” of the cross-section alongthe line I-I. However, for simplicity, the respective sources of theMOSFETs Q2 and Q3 shown in FIG. 10, as well as the gate of Q2, are notvisible in FIG. 11A, as they lie along the same axis (i.e.,perpendicular to the plane of the figure) as the shared drain (if shownin FIG. 11A, these elements would unduly complicate the compositecross-sectional view of FIG. 11A).

Above the substrate, gate oxide, and polysilicon layers shown in FIG.11A, a number of additional layers are provided to establish electricalconnections to the various pixel components, including alternating metallayers and oxide layers through which conductive vias are formed.Pursuant to the example of a 4-Metal CMOS process, these layers arelabeled in FIG. 11A as “Contact,” “Metal1,” “Via1,” “Metal2,” “Via2,”“Metal3,” “Via3,” and “Metal4.” (Note that more or fewer metal layersmay be employed.) To facilitate an understanding particularly of theISFET electrical connections, the composite cross-sectional view of FIG.11A shows additional elements of the pixel fabrication on the right sideof the top view of FIG. 10 between the lines II-II and III-III. Withrespect to the ISFET electrical connections, the topmost metal layer 304corresponds to the ISFETs sensitive area 178, above which is disposed ananalyte-sensitive passivation layer 172. The topmost metal layer 304,together with the ISFET polysilicon gate 164 and the interveningconductors 306, 308, 312, 316, 320, 326 and 338, form the ISFETs“floating gate” structure 170, in a manner similar to that discussedabove in connection with a conventional ISFET design shown in FIG. 1. Anelectrical connection to the ISFETs drain is provided by the conductors340, 328, 318, 314 and 310 coupled to the line 116 ₁. The ISFETs sourceis coupled to the shared drain of the MOSFETs Q2 and Q3 via theconductors 334 and 336 and the conductor 324 (which lies along the lineI-I in FIG. 10). The body connections 162 to the n-well 154 areelectrically coupled to a metal conductor 322 around the perimeter ofthe pixel on the “Metal1” layer via the conductors 330 and 332.

As indicated above, FIGS. 12A through 12L provide top views of each ofthe fabrication layers shown in FIG. 11A (the respective images of FIGS.12A through 12L are superimposed one on top of another to create thepixel chip layout design shown in FIG. 10). In FIG. 12, thecorrespondence between the lettered top views of respective layers andthe cross-sectional view of FIG. 11A is as follows: A) n-type well 154;B) Implant; C) Diffusion; D) polysilicon gates 164 (ISFET) and 166(MOSFETs Q2 and Q3); E) contacts; F) Metal1; G) Via1; H) Metal2; I)Via2; J) Metal3; K) Via3; L) Metal4 (top electrode contacting ISFETgate). The various reference numerals indicated in FIGS. 12A through 12Lcorrespond to the identical features that are present in the compositecross-sectional view of FIG. 11A.

At least in some applications, pixel capacitance may be a salientparameter for some type of analyte measurements. Accordingly, in anotherembodiment related to pixel layout and design, various via and metallayers may be reconfigured so as to at least partially mitigate thepotential for parasitic capacitances to arise during pixel operation.For example, in one such embodiment, pixels are designed such that thereis a greater vertical distance between the signal lines 112 ₁, 114 ₁,116 ₁ and 118 ₁, and the topmost metal layer 304 constituting thefloating gate structure 170.

In the embodiment described immediately above, with reference again toFIG. 11A, it may be readily observed that the topmost metal layer 304 isformed in the Metal4 layer (also see FIG. 12L), and the signal lines 112₁, 114 ₁, and 116 ₁ are formed in the Metal3 layer (also see FIG. 12J).Also, while not visible in the view of FIG. 11A, it may be observed fromFIG. 12H that the signal line 118 ₁ is formed in the Metal2 layer. Asone or more of these signals may be grounded from time to time duringarray operation, a parasitic capacitance may arise between any one ormore of these signal lines and metal layer 304. By increasing a distancebetween these signal lines and the metal layer 304, such parasiticcapacitance may be reduced.

To this end, in another embodiment some via and metal layers arereconfigured such that the signal lines 112 ₁, 114 ₁, 116 ₁ and 118 ₁are implemented in the Metal1 and Metal2 layers, and the Metal3 layer isused only as a jumper between the Metal2 layer component of the floatinggate structure 170 and the topmost metal layer 304, thereby ensuring agreater distance between the signal lines and the metal layer 304. FIG.10-1 illustrates a top view of a such a chip layout design for a clusterof four neighboring pixels of an chemFET array shown in FIG. 9, with oneparticular pixel 105 ₁ identified and labeled. FIG. 11A-1 shows acomposite cross-sectional view of neighboring pixels, along the line I-Iof the pixel 105 ₁ shown in FIG. 10-1, including additional elementsbetween the lines II-II, illustrating a layer-by-layer view of the pixelfabrication, and FIGS. 12-1A through 12-1L provide top views of each ofthe fabrication layers shown in FIG. 11A-1 (the respective images ofFIGS. 12-1A through 12-1L are superimposed one on top of another tocreate the pixel chip layout design shown in FIG. 10-1).

In FIG. 10-1, it may be observed that the pixel top view layout isgenerally similar to that shown in FIG. 10. For example, in the topview, the ISFET 150 generally occupies the right center portion of eachpixel, and the MOSFETs Q2 and Q3 generally occupy the left centerportion of the pixel illustration. Many of the component labels includedin FIG. 10 are omitted from FIG. 10-1 for clarity, although the ISFETpolysilicon gate 164 is indicated in the pixel 105 ₁ for orientation.FIG. 10-1 also shows the four lines (112 ₁, 114 ₁, 116 ₁ and 118 ₁)required to operate the pixel. One noteworthy difference between FIG. 10and FIG. 10-1 relates to the metal conductor 322 (located on the Metal1layer) which provides an electrical connection to the body region 162;namely, in FIG. 10, the conductor 322 surrounds a perimeter of thepixel, whereas in FIG. 10-1, the conductor 322 does not completelysurround a perimeter of the pixel but includes discontinuities 727.These discontinuities 727 permit the line 118 ₁ to also be fabricated onthe Metal1 layer and traverse the pixel to connect to neighboring pixelsof a row.

With reference now to the cross-sectional view of FIG. 11A-1, threeadjacent pixels are shown in cross-section, with the center pixelcorresponding to the pixel 105 ₁ in FIG. 10-1 for purposes ofdiscussion. As in the embodiment of FIG. 11A, all of the FET componentsof the pixel 105 ₁ are fabricated as p-channel FETs in the single n-typewell 154. Additionally, as in FIG. 11A, in the composite cross-sectionalview of FIG. 11A-1 the highly doped p-type region 159 is also visible(lying along the line I-I in FIG. 10-1), corresponding to the shareddrain (D) of the MOSFETs Q2 and Q3. For purposes of illustration, thepolysilicon gate 166 of the MOSFET Q3 also is visible in FIG. 11A-1,although this gate does not lie along the line I-I in FIG. 10-1, butrather “behind the plane” of the cross-section along the line I-I.However, for simplicity, the respective sources of the MOSFETs Q2 and Q3shown in FIG. 10-1, as well as the gate of Q2, are not visible in FIG.11A-1, as they lie along the same axis (i.e., perpendicular to the planeof the figure) as the shared drain. Furthermore, to facilitate anunderstanding of the ISFET floating gate electrical connections, thecomposite cross-sectional view of FIG. 11A-1 shows additional elementsof the pixel fabrication between the lines II-II of FIG. 10-1.

More specifically, as in the embodiment of FIG. 11A, the topmost metallayer 304 corresponds to the ISFETs sensitive area 178, above which isdisposed an analyte-sensitive passivation layer 172. The topmost metallayer 304, together with the ISFET polysilicon gate 164 and theintervening conductors 306, 308, 312, 316, 320, 326 and 338, form theISFETs floating gate structure 170. However, unlike the embodiment ofFIG. 11A, an electrical connection to the ISFETs drain is provided bythe conductors 340, 328, and 318, coupled to the line 116 ₁ which isformed in the Metal2 layer rather than the Metal3 layer. Additionally,the lines 112 ₁ and 114 ₁ also are shown in FIG. 11A-1 as formed in theMetal2 layer rather than the Metal3 layer. The configuration of theselines, as well as the line 118 ₁, may be further appreciated from therespective images of FIGS. 12-1A through 12-1L (in which thecorrespondence between the lettered top views of respective layers andthe cross-sectional view of FIG. 11A-1 is the same as that described inconnection with FIGS. 12A-12L); in particular, it may be observed inFIG. 12-1F that the line 118 ₁, together with the metal conductor 322,is formed in the Metal1 layer, and it may be observed that the lines 112₁, 114 ₁ and 116 ₁ are formed in the Metal2 layer, leaving only thejumper 308 of the floating gate structure 170 in the Metal3 layer shownin FIG. 12-1J.

Accordingly, by consolidating the signal lines 112 ₁, 114 ₁, 116 ₁ and118 ₁ to the Metal1 and Metal2 layers and thereby increasing thedistance between these signal lines and the topmost layer 304 of thefloating gate structure 170 in the Metal4 layer, parasitic capacitancesin the ISFET may be at least partially mitigated. It should beappreciated that this general concept (e.g., including one or moreintervening metal layers between signal lines and topmost layer of thefloating gate structure) may be implemented in other fabricationprocesses involving greater numbers of metal layers. For example,distance between pixel signal lines and the topmost metal layer may beincreased by adding additional metal layers (more than four total metallayers) in which only jumpers to the topmost metal layer are formed inthe additional metal layers. In particular, a six-metal-layerfabrication process may be employed, in which the signal lines arefabricated using the Metal1 and Metal2 layers, the topmost metal layerof the floating gate structure is formed in the Metal6 layer, andjumpers to the topmost metal layer are formed in the Metal3, Metal4 andMetal5 layers, respectively (with associated vias between the metallayers). In another exemplary implementation based on a six-metal-layerfabrication process, the general pixel configuration shown in FIGS. 10,11A, and 12A-12L may be employed (signal lines on Metal2 and Metal 3layers), in which the topmost metal layer is formed in the Metal6 layerand jumpers are formed in the Metal4 and Metal5 layers, respectively.

In yet another aspect relating to reduced capacitance, a dimension “f”of the topmost metal layer 304 (and thus the ISFET sensitive area 178)may be reduced so as to reduce cross-capacitance between neighboringpixels. As may be observed in FIG. 11A-1 (and as discussed further belowin connection with other embodiments directed to well fabrication abovean ISFET array), the well 725 may be fabricated so as to have a taperedshape, such that a dimension “g” at the top of the well is smaller thanthe pixel pitch “e” but yet larger than a dimension “f” at the bottom ofthe well. Based on such tapering, the topmost metal layer 304 also maybe designed with the dimension “f” rather than the dimension “g” so asto provide for additional space between the top metal layers ofneighboring pixels. In some illustrative non-limiting implementations,for pixels having a dimension “e” on the order of 9 micrometers thedimension “f” may be on the order of 6 micrometers (as opposed to 7micrometers, as discussed above), and for pixels having a dimension “e”on the order of 5 micrometers the dimension “f” may be on the order of3.5 micrometers.

Thus, the pixel chip layout designs respectively shown in FIGS. 10, 11A,and 12A through 12L, and FIGS. 10-1, 11A-1, and 12-1A through 12-1L,illustrate that according to various embodiments FET devices of a sametype may be employed for all components of a pixel, and that allcomponents may be implemented in a single well. This dramaticallyreduces the area required for the pixel, thereby facilitating increasedpixel density in a given area.

In one exemplary implementation, the gate oxide 165 for the ISFET may befabricated to have a thickness on the order of approximately 75Angstroms, giving rise to a gate oxide capacitance per unit area C_(ox)of 4.5 fF/μm². Additionally, the polysilicon gate 164 may be fabricatedwith dimensions corresponding to a channel width W of 1.2 μm and achannel length L of from 0.35 to 0.6 μm (i.e., W/L ranging fromapproximately 2 to 3.5), and the doping of the region 160 may beselected such that the carrier mobility for the p-channel is 190 cm²/V·s(i.e., 1.9E10 μm²/V·s). From Eq. (2) above, this results in an ISFETtransconductance parameter β on the order of approximately 170 to 300μA/V². In other aspects of this exemplary implementation, the analogsupply voltage VDDA is 3.3 Volts, and VB1 and VB2 are biased so as toprovide a constant ISFET drain current I_(Dj) on the order of 5 μA (insome implementations, VB1 and VB2 may be adjusted to provide draincurrents from approximately 1 μA to 20 μA). Additionally, the MOSFET Q6(see bias/readout circuitry 110 j in FIG. 9) is sized to have a channelwidth to length ratio (e.g., W/L of approximately 50) such that thevoltage across Q6, given I_(Dj) of 5 μA, is 800 mV (i.e., V_(DSj)=800mV). From Eq. (3), based on these exemplary parameters, this providesfor pixel output voltages V_(Sj) over a range of approximately 0.5 to2.5 Volts for ISFET threshold voltage changes over a range ofapproximately 0 to 2 Volts.

With respect to the analyte-sensitive passivation layer 172 shown inFIG. 11A, in exemplary CMOS implementations the passivation layer may besignificantly sensitive to the concentration of various ion species,including hydrogen, and may include silicon nitride (Si₃N₄) and/orsilicon oxynitride (Si₂N₂O). In conventional CMOS processes, apassivation layer may be formed by one or more successive depositions ofthese materials, and is employed generally to treat or coat devices soas to protect against contamination and increase electrical stability.The material properties of silicon nitride and silicon oxynitride aresuch that a passivation layer comprising these materials providesscratch protection and serves as a significant barrier to the diffusionof water and sodium, which can cause device metallization to corrodeand/or device operation to become unstable. A passivation layerincluding silicon nitride and/or silicon oxynitride also providesion-sensitivity in ISFET devices, in that the passivation layer containssurface groups that may donate or accept protons from an analytesolution with which they are in contact, thereby altering the surfacepotential and the device threshold voltage V_(TH), as discussed above inconnection with FIGS. 1 and 2A.

For CMOS processes involving aluminum as the metal (which has a meltingpoint of approximately 650 degrees Celsius), a silicon nitride and/orsilicon oxynitride passivation layer generally is formed viaplasma-enhanced chemical vapor deposition (PECVD), in which a glowdischarge at 250-350 degrees Celsius ionizes the constituent gases thatform silicon nitride or silicon oxynitride, creating active species thatreact at the wafer surface to form a laminate of the respectivematerials. In one exemplary process, a passivation layer having athickness on the order of approximately 1.0 to 1.5 μm may be formed byan initial deposition of a thin layer of silicon oxynitride (on theorder of 0.2 to 0.4 μm) followed by a slighting thicker deposition ofsilicon oxynitride (on the order of 0.5 μm) and a final deposition ofsilicon nitride (on the order of 0.5 μm). Because of the low depositiontemperature involved in the PECVD process, the aluminum metallization isnot adversely affected.

However, while a low-temperature PECVD process provides adequatepassivation for conventional CMOS devices, the low-temperature processresults in a generally low-density and somewhat porous passivationlayer, which in some cases may adversely affect ISFET threshold voltagestability. In particular, during ISFET device operation, a low-densityporous passivation layer over time may absorb and become saturated withions from the solution, which may in turn cause an undesirabletime-varying drift in the ISFETs threshold voltage V_(TH), makingaccurate measurements challenging.

In view of the foregoing, in one embodiment a CMOS process that usestungsten metal instead of aluminum may be employed to fabricate ISFETarrays according to the present disclosure. The high melting temperatureof Tungsten (above 3400 degrees Celsius) permits the use of a highertemperature low pressure chemical vapor deposition (LPCVD) process(e.g., approximately 700 to 800 degrees Celsius) for a silicon nitrideor silicon oxynitride passivation layer. The LPCVD process typicallyresults in significantly more dense and less porous films for thepassivation layer, thereby mitigating the potentially adverse effects ofion absorption from the analyte solution leading to ISFET thresholdvoltage drift.

In yet another embodiment in which an aluminum-based CMOS process isemployed to fabricate ISFET arrays according to the present disclosure,the passivation layer 172 shown in FIG. 11A may comprise additionaldepositions and/or materials beyond those typically employed in aconventional CMOS process. For example, the passivation layer 172 mayinclude initial low-temperature plasma-assisted depositions (PECVD) ofsilicon nitride and/or silicon oxynitride as discussed above; forpurposes of the present discussion, these conventional depositions areillustrated in FIG. 11A as a first portion 172A of the passivation layer172. In one embodiment, following the first portion 172A, one or moreadditional passivation materials are disposed to form at least a secondportion 172B to increase density and reduce porosity of (and absorptionby) the overall passivation layer 172. While one additional portion 172Bis shown primarily for purposes of illustration in FIG. 11A, it shouldbe appreciated that the disclosure is not limited in this respect, asthe overall passivation layer 172 may comprise two or more constituentportions, in which each portion may comprise one or morelayers/depositions of same or different materials, and respectiveportions may be configured similarly or differently. Regardless of thespecific materials, the passivation layer(s) provide chemical isolationbetween the analyte and the circuitry.

Examples of materials suitable for the second portion 172B (or otheradditional portions) of the passivation layer 172 include, but are notlimited to, silicon nitride, silicon oxynitride, aluminum oxide (Al₂O₃),tantalum oxide (Ta₃O₅), tin oxide (SnO₂) and silicon dioxide (SiO₂). Inone aspect, the second portion 172B (or other additional portions) maybe deposited via a variety of relatively low-temperature processesincluding, but not limited to, RF sputtering, DC magnetron sputtering,thermal or e-beam evaporation, and ion-assisted depositions. In anotheraspect, a pre-sputtering etch process may be employed, prior todeposition of the second portion 172B, to remove any native oxideresiding on the first portion 172A (alternatively, a reducingenvironment, such as an elevated temperature hydrogen environment, maybe employed to remove native oxide residing on the first portion 172A).In yet another aspect, a thickness of the second portion 172B may be onthe order of approximately 0.04 μm to 0.06 μm (400 to 600 Angstroms) anda thickness of the first portion may be on the order of 1.0 to 1.5 μm,as discussed above. In some exemplary implementations, the first portion172A may include multiple layers of silicon oxynitride and siliconnitride having a combined thickness of 1.0 to 1.5 μm, and the secondportion 172B may include a single layer of either aluminum oxide ortantalum oxide having a thickness of approximately 400 to 600 Angstroms.Again, it should be appreciated that the foregoing exemplary thicknessesare provided primarily for purposes of illustration, and that thedisclosure is not limited in these respects.

Thus it is to be understood that the chemFET arrays described herein maybe used to detect and/or measure various analytes and, by doing so, maymonitor a variety of reactions and/or interactions. It is also to beunderstood that the discussion herein relating to hydrogen ion detection(in the form of a pH change) is for the sake of convenience and brevityand that static or dynamic levels/concentrations of other analytes(including other ions) can be substituted for hydrogen in thesedescriptions. In particular, sufficiently fast concentration changes ofany one or more of various ion species present in the analyte may bedetected via the transient or dynamic response of a chemFET, asdiscussed above in connection with FIG. 2A. As also discussed above inconnection with the Site-Dissociation (or Site-Binding) model for theanalyte/passivation layer interface, it should be appreciated thatvarious parameters relating to the equilibrium reactions at theanalyte/passivation layer interface (e.g., rate constants for forwardand backward equilibrium reactions, total number of protondonor/acceptor sites per unit area on the passivation layer surface,intrinsic buffering capacity, pH at point of zero charge) are materialdependent properties and thus are affected by the choice of materialsemployed for the passivation layer.

The chemFETs, including ISFETs, described herein are capable ofdetecting any analyte that is itself capable of inducing a change inelectric field when in contact with or otherwise sensed or detected bythe chemFET surface. The analyte need not be charged in order to bedetected by the sensor. For example, depending on the embodiment, theanalyte may be positively charged (i.e., a cation), negatively charged(i.e., an anion), zwitterionic (i.e., capable of having two equal andopposite charges but being neutral overall), and polar yet neutral. Thislist is not intended as exhaustive as other analyte classes as well asspecies within each class will be readily contemplated by those ofordinary skill in the art based on the disclosure provided herein.

In the broadest sense of the invention, the passivation layer may or maynot be coated and the analyte may or may not interact directly with thepassivation layer.

Passivation Layer Specificity

In some embodiments, the passivation layer and/or the layers and/ormolecules coated thereon dictate the analyte specificity of the arrayreadout.

Detection of hydrogen ions, and other analytes as determined by theinvention, can be carried out using a passivation layer made of siliconnitride (Si₃N₄), silicon oxynitride (Si₂N₂O), silicon oxide (SiO₂),aluminum oxide (Al₂O₃), tantalum pentoxide (Ta₂O₅), tin oxide or stannicoxide (SnO₂), and the like.

The passivation layer can also detect other ion species directlyincluding but not limited to calcium, potassium, sodium, iodide,magnesium, chloride, lithium, lead, silver, cadmium, nitrate, phosphate,dihydrogen phosphate, and the like.

In some embodiments, the passivation layer is coated with a receptor forthe analyte of interest. Preferably, the receptor binds selectively tothe analyte of interest or in some instances to a class of agents towhich the analyte belongs. As used herein, a receptor that bindsselectively to an analyte is a molecule that binds preferentially tothat analyte (i.e., its binding affinity for that analyte is greaterthan its binding affinity for any other analyte). Its binding affinityfor the analyte of interest may be 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold,30-fold, 40-fold, 50-fold, 100-fold or more than its binding affinityfor any other analyte. In addition to its relative binding affinity, thereceptor must also have an absolute binding affinity that issufficiently high to efficiently bind the analyte of interest (i.e., itmust have a sufficient sensitivity). Receptors having binding affinitiesin the picomolar to micromolar range are suitable. Preferably suchinteractions are reversible.

The receptor may be of any nature (e.g., chemical, nucleic acid,peptide, lipid, combinations thereof and the like). In such embodiments,the analyte too may be of any nature provided there exists a receptorthat binds to it selectively and in some instances specifically. It isto be understood however that the invention further contemplatesdetection of analytes in the absence of a receptor. An example of thisis the detection of PPi and Pi by the passivation layer in the absenceof PPi or Pi receptors.

In one aspect, the invention contemplates receptors that are ionophores.As used herein, an ionophore is a molecule that binds selectively to anionic species, whether anion or cation. In the context of the invention,the ionophore is the receptor and the ion to which it binds is theanalyte. Ionophores of the invention include art-recognized carrierionophores (i.e., small lipid-soluble molecules that bind to aparticular ion) derived from microorganisms. Various ionophores arecommercially available from sources such as Calbiochem.

Detection of some ions can be accomplished through the use of thepassivation layer itself or through the use of receptors coated onto thepassivation layer. For example, potassium can be detected selectivelyusing polysiloxane, valinomycin, or salinomycin; sodium can be detectedselectively using monensin, nystatin, or SQI-Pr; calcium can be detectedselectively using ionomycin, calcimycine (A23187), or CA 1001 (ETH1001).

Receptors able to bind more than one ion can also be used in someinstances. For example, beauvericin can be used to detect calcium and/orbarium ions, nigericin can be used to detect potassium, hydrogen and/orlead ions, and gramicidin can be used to detect hydrogen, sodium and/orpotassium ions. One of ordinary skill in the art will recognize thatthese compounds can be used in applications in which single ionspecificity is not required or in which it is unlikely (or impossible)that other ions which the compounds bind will be present or generated.Similarly, receptors that bind multiple species of a particular genusmay also be useful in some embodiments including those in which only onespecies within the genus will be present or in which the method does notrequire distinction between species.

As another example, receptors for neurotoxins are described in SimonianElectroanalysis 2004, 16: 1896-1906.

Passivation Layer and PPi Receptors

In other embodiments, including but not limited to nucleic acidsequencing applications, receptors that bind selectively to PPi can beused. Examples of PPi receptors include those compounds shown in FIGS.11B(1)-(3) (compounds 1-10). Compound 1 is described in Angew, Chem Int(Ed 2004) 43:4777-4780 and US 2005/0119497 A1 and is referred to asp-naphthyl-bis[(bis(2-pyridylmethyl)amino)methyl]phenol. Compound 2 isdescribed in J Am Chem Soc 2003 125:7752-7753 and US 2005/0119497 A1 andis referred to asp-(p-nitrophenylazo)-bis[(bis(2-pyridylmethyl-1)amino)methyl]phenol (orits dinuclear Zn complex). Synthesis schemes for compounds 1 and 2 areshown provided in US 2005/0119497 A1. Compound 3 is described in by Leeet al. Organic Letters 2007 9(2):243-246, and Sensors and Actuators B1995 29:324-327. Compound 4 is described in Angew, Chem Int (Ed 2002)41(20):3811-3814. Exemplary syntheses for compounds 7, 8 and 9 are shownin FIGS. 11C(1)-(3). Compound 5 is described in WO 2007/002204 and isreferred to therein as bis-Zn²⁺-dipicolylamine (Zn²⁺-DPA). Compound 6 isillustrated in FIG. 11B(3) bound to PPi. (McDonough et al. Chem. Commun.2006 2971-2973.) Attachment of compound 7 to a metal oxide surface isshown in FIG. 11E.

Passivation Layer—Receptor Binding

Receptors may be attached to the passivation layer covalently ornon-covalently. Covalent attachment of a receptor to the passivationlayer may be direct or indirect (e.g., through a linker). FIGS. 11D(1)and (2) illustrate the use of silanol chemistry to covalently bindreceptors to the passivation layer. Receptors may be immobilized on thepassivation layer using for example aliphatic primary amines (bottomleft panel) or aryl isothiocyanates (bottom right panel). In these andother embodiments, the passivation layer which itself may be comprisedof silicon nitride, aluminum oxide, silicon oxide, tantalum pentoxide,or the like, is bonded to a silanation layer via its reactive surfacegroups. For greater detail on silanol chemistry for covalent attachmentto the FET surface, reference can be made to at least the followingpublications: for silicon nitride, see Sensors and Actuators B 199529:324-327, Jpn J Appl Phys 1999 38:3912-3917 and Langmuir 200521:395-402; for silicon oxide, see Protein Sci 1995 4:2532-2544 and AmBiotechnol Lab 2002 20(7):16-18; and for aluminum oxide, see Colloidsand Surfaces 1992 63:1-9, Sensors and Actuators B 2003 89:40-47, andBioconjugate Chem 1997 8:424-433. The receptor is then conjugated to thesilanation layer reactive groups. This latter binding can occur directlyor indirectly through the use of a bifunctional linker, as illustratedin FIGS. 11D(1) and (2).

A bifunctional linker is a compound having at least two reactive groupsto which two entities may be bound. In some instances, the reactivegroups are located at opposite ends of the linker. In some embodiments,the bifunctional linker is a universal bifunctional linker such as thatshown in FIGS. 11D(1) and (2). A universal linker is a linker that canbe used to link a variety of entities. It should be understood that thechemistries shown in FIGS. 11D(1) and (2) are meant to be illustrativeand not limiting.

The bifunctional linker may be a homo-bifunctional linker or ahetero-bifunctional linker, depending upon the nature of the moleculesto be conjugated. Homo-bifunctional linkers have two identical reactivegroups. Hetero-bifunctional linkers are have two different reactivegroups. Various types of commercially available linkers are reactivewith one or more of the following groups: primary amines, secondaryamines, sulphydryls, carboxyls, carbonyls and carbohydrates. Examples ofamine-specific linkers are bis(sulfosuccinimidyl) suberate,bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate,disuccinimidyl tartarate, dimethyl adipimate.2HCl, dimethylpimelimidate.2HCl, dimethyl suberimidate.2HCl, and ethyleneglycolbis-[succinimidyl-[succinate]]. Linkers reactive with sulfhydrylgroups include bismaleimidohexane,1,4-di-[3′-(2′-pyridyldithio)-propionamido)]butane,1-[p-azidosalicylamido]-4-[iodoacetamido]butane, andN-[4-(p-azidosalicylamido) butyl]-3′-[2′-pyridyldithio]propionamide.Linkers preferentially reactive with carbohydrates include azidobenzoylhydrazine. Linkers preferentially reactive with carboxyl groups include4-[p-azidosalicylamido]butylamine.

Heterobifunctional linkers that react with amines and sulfhydrylsinclude N-succinimidyl-3-[2-pyridyldithio]propionate, succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Heterobifunctionallinkers that react with carboxyl and amine groups include1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.Heterobifunctional linkers that react with carbohydrates and sulfhydrylsinclude 4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide.2HCl,4-(4-N-maleimidophenyl)-butyric acid hydrazide.2HCl, and3-[2-pyridyldithio]propionyl hydrazide.

Alternatively, receptors may be non-covalently coated onto thepassivation layer. Non-covalent deposition of the receptor onto thepassivation layer may involve the use of a polymer matrix. The polymermay be naturally occurring or non-naturally occurring and may be of anytype including but not limited to nucleic acid (e.g., DNA, RNA, PNA,LNA, and the like, or mimics, derivatives, or combinations thereof),amino acid (e.g., peptides, proteins (native or denatured), and thelike, or mimics, derivatives, or combinations thereof, lipids,polysaccharides, and functionalized block copolymers. The receptor maybe adsorbed onto and/or entrapped within the polymer matrix. The natureof the polymer will depend on the nature of the receptor being usedand/or analyte being detected.

Alternatively, the receptor may be covalently conjugated or crosslinkedto the polymer (e.g., it may be “grafted” onto a functionalizedpolymer).

An example of a suitable peptide polymer is poly-lysine (e.g.,poly-L-lysine). Examples of other polymers include block copolymers thatcomprise polyethylene glycol (PEG), polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitrocelluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, polyvinylchloride, polystyrene, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate), poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, polyanhydrides, poly(styrene-b-isobutylene-b-styrene) (SIBS) block copolymer, ethylene vinylacetate, poly(meth)acrylic acid, polymers of lactic acid and glycolicacid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(buticacid), poly(valeric acid), and poly(lactide-cocaprolactone), and naturalpolymers such as alginate and other polysaccharides including dextranand cellulose, collagen, albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof, and chemical derivatives thereof including substitutions and/oradditions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art.

Trapped Charge

Another issue that relates to ISFET threshold voltage stability and/orpredictability involves trapped charge that may accumulate (especially)on metal layers of CMOS-fabricated devices as a result of variousprocessing activities during or following array fabrication (e.g.,back-end-of-line processing such as plasma metal etching, wafercleaning, dicing, packaging, handling, etc.). In particular, withreference to FIG. 11A, trapped charge may in some instances accumulateon one or more of the various conductors 304, 306, 308, 312, 316, 320,326, 338, and 164 constituting the ISFETs floating gate structure 170.This phenomenon also is referred to in the relevant literature as the“antenna effect.”

One opportunity for trapped charge to accumulate includes plasma etchingof the topmost metal layer 304. Other opportunities for charge toaccumulate on one or more conductors of the floating gate structure orother portions of the FETs includes wafer dicing, during which theabrasive process of a dicing saw cutting through a wafer generatesstatic electricity, and/or various post-processing waferhandling/packaging steps, such as die-to-package wire bonding, where insome cases automated machinery that handles/transports wafers may besources of electrostatic discharge (ESD) to conductors of the floatinggate structure. If there is no connection to the silicon substrate (orother semi-conductor substrate) to provide an electrical path to bleedoff such charge accumulation, charge may build up to the point ofcausing undesirable changes or damage to the gate oxide 165 (e.g.,charge injection into the oxide, or low-level oxide breakdown to theunderlying substrate). Trapped charge in the gate oxide or at the gateoxide-semiconductor interface in turn can cause undesirable and/orunpredictable variations in ISFET operation and performance, such asfluctuations in threshold voltage.

In view of the foregoing, other inventive embodiments of the presentdisclosure are directed to methods and apparatus for improving ISFETperformance by reducing trapped charge or mitigating the antenna effect.In one embodiment, trapped charge may be reduced after a sensor arrayhas been fabricated, while in other embodiments the fabrication processitself may be modified to reduce trapped charge that could be induced bysome conventional process steps. In yet other embodiments, both “duringfabrication” and “post fabrication” techniques may be employed incombination to reduce trapped charge and thereby improve ISFETperformance.

With respect to alterations to the fabrication process itself to reducetrapped charge, in one embodiment the thickness of the gate oxide 165shown in FIG. 11A may be particularly selected so as to facilitatebleeding of accumulated charge to the substrate; in particular, athinner gate oxide may allow a sufficient amount of built-up charge topass through the gate oxide to the substrate below without becomingtrapped. In another embodiment based on this concept, a pixel may bedesigned to include an additional “sacrificial” device, i.e., anothertransistor having a thinner gate oxide than the gate oxide 165 of theISFET. The floating gate structure of the ISFET may then be coupled tothe gate of the sacrificial device such that it serves as a “chargebleed-off transistor.” Of course, it should be appreciated that sometrade-offs for including such a sacrificial device include an increasein pixel size and complexity.

In another embodiment, the topmost metal layer 304 of the ISFETsfloating gate structure 170 shown in FIG. 11A may be capped with adielectric prior to plasma etching to mitigate trapped charge. Asdiscussed above, charge accumulated on the floating gate structure mayin some cases be coupled from the plasma being used for metal etching.Typically, a photoresist is applied over the metal to be etched and thenpatterned based on. the desired geometry for the underlying metal. Inone exemplary implementation, a capping dielectric layer (e.g., anoxide) may be deposited over the metal to be etched, prior to theapplication of the photoresist, to provide an additional barrier on themetal surface against charge from the plasma etching process. In oneaspect, the capping dielectric layer may remain behind and form aportion of the passivation layer 172.

In yet another embodiment, the metal etch process for the topmost metallayer 304 may be modified to include wet chemistry or ion-beam millingrather than plasma etching. For example, the metal layer 304 could beetched using an aqueous chemistry, selective to the underlyingdielectric (e.g., see website for Transene relating to aluminum, whichis hereby incorporated herein by reference). Another alternativeapproach employs ion-milling rather than plasma etching for the metallayer 304. Ion-milling is commonly used to etch materials that cannot bereadily removed using conventional plasma or wet chemistries. Theion-milling process does not employ an oscillating electric field asdoes a plasma, so that charge build-up does not occur in the metallayer(s). Yet another metal etch alternative involves optimizing theplasma conditions so as to reduce the etch rate (i.e. less powerdensity).

In yet another embodiment, architecture changes may be made to the metallayer to facilitate complete electrical isolation during definition ofthe floating gate. In one aspect, designing the metal stack-up so thatthe large area ISFET floating gate is not connected to anything duringits final definition may require a subsequent metal layer serving as a“jumper” to realize the electrical connection to the floating gate ofthe transistor. This “jumper” connection scheme prevents charge flowfrom the large floating gate to the transistor. This method may beimplemented as follows (M=metal layer): i) M1 contacting Poly gateelectrode; ii) M2 contacting M1; iii) M3 defines floating gate andseparately connects to M2 with isolated island; iv) M4 jumper, havingvery small area being etched over the isolated islands and connectionsto floating gate M3, connects the M3 floating gate to the M1/M2/M3 stackconnected to the Poly gate immediately over the transistor active area;and v) M3 to M4 interlayer dielectric is removed only over the floatinggate so as to expose the bare M3 floating gate. In the method outlinedimmediately above, step v) need not be done, as the ISFET architectureaccording to some embodiments discussed above leaves the M4 passivationin place over the M4 floating gate. In one aspect, removal maynonetheless improve ISFET performance in other ways (i.e. sensitivity).In any case, the final sensitive passivation layer may be a thinsputter-deposited ion-sensitive metal-oxide layer. It should beappreciated that the over-layer jumpered architecture discussed abovemay be implemented in the standard CMOS fabrication flow to allow any ofthe first three metal layers to be used as the floating gates (i.e. M1,M2 or M3).

With respect to post-fabrication processes to reduce trapped charge, inone embodiment a “forming gas anneal” may be employed as apost-fabrication process to mitigate potentially adverse effects oftrapped charge. In a forming gas anneal, CMOS-fabricated ISFET devicesare heated in a hydrogen and nitrogen gas mixture. The hydrogen gas inthe mixture diffuses into the gate oxide 165 and neutralizes certainforms of trapped charges. In one aspect, the forming gas anneal need notnecessarily remove all gate oxide damage that may result from trappedcharges; rather, in some cases, a partial neutralization of some trappedcharge is sufficient to significantly improve ISFET performance. Inexemplary annealing processes according to the present disclosure,ISFETs may be heated for approximately 30 to 60 minutes at approximately400 to 425 degrees Celsius in a hydrogen/nitrogen mixture that includes10% to 15% hydrogen. In one particular implementation, annealing at 425degrees Celsius at 30 minutes in a hydrogen/nitrogen mixture thatincludes 10% hydrogen is observed to be particularly effective atimproving ISFET performance. For aluminum CMOS processes, thetemperature of the anneal should be kept at or below 450 degrees Celsiusto avoid damaging the aluminum metallurgy. In another aspect of anannealing process according to the present disclosure, the forming gasanneal is performed after wafers of fabricated ISFET arrays are diced,so as to ensure that damage due to trapped charge induced by the dicingprocess itself, and/or other pre-dicing processing steps (e.g., plasmaetching of metals) may be effectively ameliorated. In yet anotheraspect, the forming gas anneal may be performed after die-to-packagewirebonding to similarly ameliorate damage due to trapped charge. Atthis point in the assembly process, a diced array chip is typically in aheat and chemical resistant ceramic package, and low-tolerancewirebonding procedures as well as heat-resistant die-to-packageadhesives may be employed to withstand the annealing procedure. Thus, inone exemplary embodiment, the invention encompasses a method formanufacturing an array of FETs, each having or coupled to a floatinggate having a trapped charge of zero or substantially zero comprising:fabricating a plurality of FETs in a common semiconductor substrate,each of a plurality of which is coupled to a floating gate; applying aforming gas anneal to the semiconductor prior to a dicing step; dicingthe semiconductor; and applying a forming gas anneal to thesemiconductor after the dicing step. Preferably, the semiconductorsubstrate comprises at least 100,000 FETs. Preferably, the plurality ofFETs are chemFETs. The method may further comprise depositing apassivation layer on the semiconductor, depositing a polymeric, glass,ion-reactively etchable or photodefineable material layer on thepassivation layer and etching the polymeric, glass ion-reactivelyetchable or photodefineable material to form an array of reactionchambers in the glass layer.

In yet other processes for mitigating potentially adverse effects oftrapped charge according to embodiments of the present disclosure, avariety of “electrostatic discharge (ESD)-sensitive protocols” may beadopted during any of a variety of wafer post-fabricationhandling/packaging steps. For example, in one exemplary process,anti-static dicing tape may be employed to hold wafer substrates inplace (e.g., during the dicing process). Also, although high-resistivity(e.g., 10 MΩ) deionized water conventionally is employed in connectionwith cooling of dicing saws, according to one embodiment of the presentdisclosure less resistive/more conductive water may be employed for thispurpose to facilitate charge conduction via the water; for example,deionized water may be treated with carbon dioxide to lower resistivityand improve conduction of charge arising from the dicing process.Furthermore, conductive and grounded die-ejection tools may be usedduring various wafer dicing/handling/packaging steps, again to provideeffective conduction paths for charge generated during any of thesesteps, and thereby reduce opportunities for charge to accumulate on oneor more conductors of the floating gate structure of respective ISFETsof an array.

In yet another embodiment involving a post-fabrication process to reducetrapped charge, the gate oxide region of an ISFET may be irradiated withUV radiation. With reference again to FIG. 11A, in one exemplaryimplementation based on this embodiment, an optional hole or window 302is included during fabrication of an ISFET array in the top metal layer304 of each pixel of the array, proximate to the ISFET floating gatestructure. This window is intended to allow UV radiation, whengenerated, to enter the ISFETs gate region; in particular, the variouslayers of the pixel 105 ₁, as shown in FIGS. 11 and 12 A-L, areconfigured such that UV radiation entering the window 302 may impinge inan essentially unobstructed manner upon the area proximate to thepolysilicon gate 164 and the gate oxide 165.

To facilitate a UV irradiation process to reduce trapped charge,materials other than silicon nitride and silicon oxynitride generallyneed to be employed in the passivation layer 172 shown in FIG. 11A, assilicon nitride and silicon oxynitride significantly absorb UVradiation. In view of the foregoing, these materials need to besubstituted with others that are appreciably transparent to UVradiation, examples of which include, but are not limited to,phososilicate glass (PSG) and boron-doped phososilicate glass (BPSG).PSG and BPSG, however, are not impervious to hydrogen and hydroxyl ions;accordingly, to be employed in a passivation layer of an ISFET designedfor pH sensitivity, PSG and BPSG may be used together with anion-impervious material that is also significantly transparent to UVradiation, such as aluminum oxide (Al₂O₃), to form the passivationlayer. For example, with reference again to FIG. 11A, PSG or BPSG may beemployed as a substitute for silicon nitride or silicon oxynitride inthe first portion 172A of the passivation layer 172, and a thin layer(e.g., 400 to 600 Angstroms) of aluminum oxide may be employed in thesecond portion 172B of the passivation layer 172 (e.g., the aluminumoxide may be deposited using a post-CMOS lift-off lithography process).

In another aspect of an embodiment involving UV irradiation, each ISFETof a sensor array must be appropriately biased during a UV irradiationprocess to facilitate reduction of trapped charge. In particular, highenergy photons from the UV irradiation, impinging upon the bulk siliconregion 160 in which the ISFET conducting channel is formed, createelectron-hole pairs which facilitate neutralization of trapped charge inthe gate oxide as current flows through the ISFETs conducting channel.To this end, an array controller, discussed further below in connectionwith FIG. 17, generates appropriate signals for biasing the ISFETs ofthe array during a UV irradiation process. In particular, with referenceagain to FIG. 9, each of the signals RowSel₁ through RowSel_(n) isgenerated so as to enable/select (i.e., turn on) all rows of the sensorarray at the same time and thereby couple all of the ISFETs of the arrayto respective controllable current sources 106 _(j) in each column. Withall pixels of each column simultaneously selected, the current from thecurrent source 106 _(j) of a given column is shared by all pixels of thecolumn. The column amplifiers 107A and 107B are disabled by removing thebias voltage VB4, and at the same time the output of the amplifier 107B,connected to the drain of each ISFET in a given column, is grounded viaa switch responsive to a control signal “UV.” Also, the common bodyvoltage V_(BODY) for all ISFETs of the array is coupled to electricalground (i.e., V_(BODY)=0 Volts) (as discussed above, during normaloperation of the array, the body bias voltage V_(BODY) is coupled to thehighest voltage potential available to the array, e.g., VDDA). In oneexemplary procedure, the bias voltage VB1 for all of the controllablecurrent sources 106 _(j) is set such that each pixel's ISFET conductsapproximately 1 μA of current. With the ISFET array thusly biased, thearray then is irradiated with a sufficient dose of UV radiation (e.g.,from an EPROM eraser generating approximately 20 milliWatts/cm² ofradiation at a distance of approximately one inch from the array forapproximately 1 hour). After irradiation, the array may be allowed torest and stabilize over several hours before use for measurements ofchemical properties such as ion concentration.

Utilizing at least one of the above-described techniques for reducingtrapped charge, we have been able to fabricate FETs floating gateshaving a trapped charge of zero or substantially zero. Thus, in someembodiments, an aspect of the invention encompasses a floating gatehaving a surface area of about 4 μm² to about 50 μm² having baselinethreshold voltage and preferably a trapped charge of zero orsubstantially zero. Preferably the FETs are chemFETs. The trapped chargeshould be kept to a level that does not cause appreciable variationsfrom FET to FET across the array, as that would limit the dynamic rangeof the devices, consistency of measurements, and otherwise adverselyaffect performance.

Array and Chip Design

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an ISFET sensor array 100 based on the column andpixel designs discussed above in connection with FIGS. 9-12, accordingto one embodiment of the present disclosure. In one aspect of thisembodiment, the array 100 includes 512 columns 102 ₁ through 102 ₅₁₂with corresponding column bias/readout circuitry 110 ₁ through 110 ₅₁₂(one for each column, as shown in FIG. 9), wherein each column includes512 geometrically square pixels 105 ₁ through 105 ₅₁₂, each having asize of approximately 9 micrometers by 9 micrometers (i.e., the array is512 columns by 512 rows). In another aspect, the entire array (includingpixels together with associated row and column select circuitry andcolumn bias/readout circuitry) may be fabricated on a semiconductor dieas an application specific integrated circuit (ASIC) having dimensionsof approximately 7 millimeters by 7 millimeters. While an array of 512by 512 pixels is shown in the embodiment of FIG. 13, it should beappreciated that arrays may be implemented with different numbers ofrows and columns and different pixel sizes according to otherembodiments, as discussed further below in connection with FIGS. 19-23.

Also, as discussed above, it should be appreciated that arrays accordingto various embodiments of the present invention may be fabricatedaccording to conventional CMOS fabrications techniques, as well asmodified CMOS fabrication techniques (e.g., to facilitate realization ofvarious functional aspects of the chemFET arrays discussed herein, suchas additional deposition of passivation materials, process steps tomitigate trapped charge, etc.) and other semiconductor fabricationtechniques beyond those conventionally employed in CMOS fabrication.Additionally, various lithography techniques may be employed as part ofan array fabrication process. For example, in one exemplaryimplementation, a lithography technique may be employed in whichappropriately designed blocks are “stitched” together by overlapping theedges of a step and repeat lithography exposures on a wafer substrate byapproximately 0.2 micrometers. In a single exposure, the maximum diesize typically is approximately 21 millimeters by 21 millimeters. Byselectively exposing different blocks (sides, top & bottoms, core, etc.)very large chips can be defined on a wafer (up to a maximum, in theextreme, of one chip per wafer, commonly referred to as “wafer scaleintegration”).

In one aspect of the array 100 shown in FIG. 13, the first and last twocolumns 102 ₁, 102 ₂, 102 ₅₁₁ and 102 ₅₁₂, as well as the first twopixels 105 ₁ and 105 ₂ and the last two pixels 105 ₅₁₁ and 105 ₅₁₂ ofeach of the columns 102 ₃ through 102 ₅₁₀ (e.g., two rows and columns ofpixels around a perimeter of the array) may be configured as “reference”or “dummy” pixels 103. With reference to FIG. 11A, for the dummy pixelsof an array, the topmost metal layer 304 of each dummy pixel's ISFET(coupled ultimately to the ISFETs polysilicon gate 164) is tied to thesame metal layer of other dummy pixels and is made accessible as aterminal of the chip, which in turn may be coupled to a referencevoltage VREF. As discussed above in connection with FIG. 9, thereference voltage VREF also may be applied to the bias/readout circuitryof respective columns of the array. In some exemplary implementationsdiscussed further below, preliminary test/evaluation data may beacquired from the array based on applying the reference voltage VREF andselecting and reading out dummy pixels, and/or reading out columns basedon the direct application of VREF to respective column buffers (e.g.,via the CAL signal), to facilitate offset determination (e.g.,pixel-to-pixel and column-to-column variances) and array calibration.

In yet another implementation of an array similar to that shown in FIG.13, rather than reserving the first and last two columns of 512 columnsand the first and last two pixels of each column of 512 pixels asreference pixels, the array may be fabricated to include an additionaltwo rows/columns of reference pixels surrounding a perimeter of a 512 by512 region of active pixels, such that the total size of the array interms of actual pixels is 516 by 516 pixels. As arrays of various sizesand configurations are contemplated by the present disclosure, it shouldbe appreciated that the foregoing concept may be applied to any of theother array embodiments discussed herein. For purposes of the discussionimmediately below regarding the exemplary array 100 shown in FIG. 13, atotal pixel count for the array of 512 by 512 pixels is considered.

In FIG. 13, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array via electrical connections (e.g., pins, metal pads) andlabeled for simplicity in block 195 as “supply and bias connections.”The array 100 of FIG. 13 also includes a row select shift register 192,two sets of column select shift registers 194 _(1,2) and two outputdrivers 198 ₁ and 198 ₂ to provide two parallel array output signals,Vout1 and Vout2, representing sensor measurements (i.e., collections ofindividual output signals generated by respective ISFETs of the array).The various power supply and bias voltages, control signals for the rowand column shift registers, and control signals for the columnbias/readout circuitry shown in FIG. 13 are provided by an arraycontroller, as discussed further below in connection with FIG. 17, whichalso reads the array output signals Vout1 and Vout2 (and other optionalstatus/diagnostic signals) from the array 100. In another aspect of thearray embodiment shown in FIG. 13, configuring the array such thatmultiple regions (e.g., multiple columns) of the array may be read atthe same time via multiple parallel array output signals (e.g., Vout1and Vout2) facilitates increased data acquisition rates, as discussedfurther below in connection with FIGS. 17 and 18. While FIG. 13illustrates an array having two column select registers and parallelarray output signals Vout1 and Vout2 to acquire data simultaneously fromtwo columns at a time, it should be appreciated that, in otherembodiments, arrays according to the present disclosure may beconfigured to have only one measurement signal output, or more than twomeasurement signal outputs; in particular, as discussed further below inconnection with FIGS. 19-23, more dense arrays according to otherinventive embodiments may be configured to have four our more parallelmeasurement signal outputs and simultaneously enable different regionsof the array to provide data via the four or more outputs.

FIG. 14 illustrates the row select shift register 192, FIG. 15illustrates one of the column select shift registers 194 ₂ and FIG. 16illustrates one of the output drivers 198 ₂ of the array 100 shown inFIG. 13, according to one exemplary implementation. As shown in FIGS. 14and 15, the row and column select shift registers are implemented as aseries of D-type flip-flops coupled to a digital circuitry positivesupply voltage VDDD and a digital supply ground VSSD. In the row andcolumn shift registers, a data signal is applied to a D-input of firstflip-flop in each series and a clock signal is applied simultaneously toa clock input of all of the flip-flops in the series. For eachflip-flop, a “Q” output reproduces the state of the D-input upon atransition (e.g., falling edge) of the clock signal. With reference toFIG. 14, the row select shift register 192 includes 512 D-typeflip-flops, in which a first flip-flop 193 receives a vertical datasignal DV and all flip-flops receive a vertical clock signal CV. A “Q”output of the first flip-flop 193 provides the first row select signalRowSel₁ and is coupled to the D-input of the next flip-flop in theseries. The Q outputs of successive flip-flops are coupled to theD-inputs of the next flip-flop in the series and provide the row selectsignals RowSel₂ through RowSel₅₁₂ with successive falling edgetransitions of the vertical clock signal CV, as discussed further belowin connection with FIG. 18. The last row select signal RowSel₅₁₂ alsomay be taken as an optional output of the array 100 as the signal LSTV(Last STage Vertical), which provides an indication (e.g., fordiagnostic purposes) that the last row of the array has been selected.While not shown explicitly in FIG. 14, each of the row select signalsRowSel₁ through RowSels₅₁₂ is applied to a corresponding inverter, theoutput of which is used to enable a given pixel in each column (asillustrated in FIG. 9 by the signals RowSel₁ through RowSel_(n) ).

Regarding the column select shift registers 194 ₁ and 194 ₂, these areimplemented in a manner similar to that of the row select shiftregisters, with each column select shift register comprising 256series-connected flip-flops and responsible for enabling readout fromeither the odd columns of the array or the even columns of the array.For example, FIG. 15 illustrates the column select shift register 194 ₂,which is configured to enable readout from all of the even numberedcolumns of the array in succession via the column select signalsColSel₂, ColSel₄, . . . ColSel₅₁₂, whereas another column select shiftregister 194 ₁ is configured to enable readout from all of the oddnumbered columns of the array in succession (via column select signalsColSel₁, ColSel₃, . . . Col Sel₅₁₁). Both column select shift registersare controlled simultaneously by the horizontal data signal DH and thehorizontal clock signal CH to provide the respective column selectsignals, as discussed further below in connection with FIG. 18. As shownin FIG. 15, the last column select signal ColSel₅₁₂ also may be taken asan optional output of the array 100 as the signal LSTH (Last STageHorizontal), which provides an indication (e.g., for diagnosticpurposes) that the last column of the array has been selected.

With reference again for the moment to FIG. 7, an implementation forarray row and column selection based on shift registers, as discussedabove in connection with FIGS. 13-15, is a significant improvement tothe row and column decoder approach employed in various prior art ISFETarray designs, including the design of Milgrew et al. shown in FIG. 7.In particular, regarding the row decoder 92 and the column decoder 94shown in FIG. 7, the complexity of implementing these components in anintegrated circuit array design increases dramatically as the size ofthe array is increased, as additional inputs to both decoders arerequired. For example, an array having 512 rows and columns as discussedabove in connection with FIG. 13 would require nine inputs (2⁹=512) perrow and column decoder if such a scheme were employed for row and columnselection; similarly, arrays having 7400 rows and 7400 columns, asdiscussed below in connection with other embodiments, would require 13inputs (2¹³=8192) per row and column decoder. In contrast, the row andcolumn select shift registers shown in FIGS. 14 and 15 require noadditional input signals as array size is increased, but ratheradditional D-type flip-flops (which are routinely implemented in a CMOSprocess). Thus, the shift register implementations shown in FIGS. 14 and15 provide an easily scalable solution to array row and columnselection.

In the embodiment of FIG. 13, the “odd” column select shift register 194₁ provides odd column select signals to an “odd” output driver 198, andthe even column select shift register 194 ₂ provides even column selectsignals to an “even” output driver 198 ₂. Both output drivers areconfigured similarly, and an example of the even output driver 198 ₂ isshown in FIG. 16. In particular, FIG. 16 shows that respective evencolumn output signals V_(COL2), V_(COL4), . . . V_(COL512) (refer toFIG. 9 for the generic column signal output V_(COLj)) are applied tocorresponding switches 191 ₂, 191 ₄, . . . 191 ₅₁₂, responsive to theeven column select signals ColSel₂, ColSel₄, . . . ColSel₅₁₂ provided bythe column select register 194 ₂, to successively couple the even columnoutput signals to the input of a buffer amplifier 199 (BUF) via a bus175. In FIG. 16, the buffer amplifier 199 receives power from an outputbuffer positive supply voltage VDDO and an output buffer supply groundVSSO, and is responsive to an output buffer bias voltage VBO0 to set acorresponding bias current for the buffer output. Given the highimpedance input of the buffer amplifier 199, a current sink 197responsive to a bias voltage VB3 is coupled to the bus 175 to provide anappropriate drive current (e.g., on the order of approximately 100 μA)for the output of the column output buffer (see the buffer amplifier 111j of FIG. 9) of a selected column. The buffer amplifier 199 provides theoutput signal Vout2 based on the selected even column of the array; atthe same time, with reference to FIG. 13, a corresponding bufferamplifier of the “odd” output driver 198 ₁ provides the output signalVout1 based on a selected odd column of the array.

In one exemplary implementation, the switches of both the even and oddoutput drivers 198 ₁ and 198 ₂ (e.g., the switches 191 ₂, 191 ₄, . . .191 ₅₁₂ shown in FIG. 16) may be implemented as CMOS-pair transmissiongates (including an n-channel MOSFET and a p-channel MOSFET; see FIG.4), and inverters may be employed so that each column select signal andits complement may be applied to a given transmission gate switch 191 toenable switching. Each switch 191 has a series resistance when enabledor “on” to couple a corresponding column output signal to the bus 175;likewise, each switch adds a capacitance to the bus 175 when the switchis off. A larger switch reduces series resistance and allows a higherdrive current for the bus 175, which generally allows the bus 175 tosettle more quickly; on the other hand, a larger switch increasescapacitance of the bus 175 when the switch is off, which in turnincreases the settling time of the bus 175. Hence, there is a trade-offbetween switch series resistance and capacitance in connection withswitch size.

The ability of the bus 175 to settle quickly following enabling ofsuccessive switches in turn facilitates rapid data acquisition from thearray. To this end, in some embodiments the switches 191 of the outputdrivers 198 ₁ and 198 ₂ are particularly configured to significantlyreduce the settling time of the bus 175. Both the n-channel and thep-channel MOSFETs of a given switch add to the capacitance of the bus175; however, n-channel MOSFETs generally have better frequency responseand current drive capabilities than their p-channel counterparts. Inview of the foregoing, some of the superior characteristics of n-channelMOSFETs may be exploited to improve settling time of the bus 175 byimplementing “asymmetric” switches in which respective sizes for then-channel MOSFET and p-channel MOSFET of a given switch are different.

For example, in one embodiment, with reference to FIG. 16, the currentsink 197 may be configured such that the bus 175 is normally “pulleddown” when all switches 191 ₂, 191 ₄, . . . 191 ₅₁₂ are open or off (notconducting). Given a somewhat limited expected signal dynamic range forthe column output signals based on ISFET measurements, when a givenswitch is enabled or on (conducting), in many instances most of theconduction is done by the n-channel MOSFET of the CMOS-pair constitutingthe switch. Accordingly, in one aspect of this embodiment, the n-channelMOSFET and the p-channel MOSFET of each switch 191 are sizeddifferently; namely, in one exemplary implementation, the n-channelMOSFET is sized to be significantly larger than the p-channel MOSFET.More specifically, considering equally-sized n-channel and p-channelMOSFETs as a point of reference, in one implementation the n-channelMOSFET may be increased to be about 2 to 2.5 times larger, and thep-channel MOSFET may be decreased in size to be about 8 to 10 timessmaller, such that the n-channel MOSFET is approximately 20 times largerthan the p-channel MOSFET. Due to the significant decrease in size ofthe p-channel MOSFET and the relatively modest increase in size of then-channel MOSFET, the overall capacitance of the switch in the off stateis notably reduced, and there is a corresponding notable reduction incapacitance for the bus 175; at the same time, due to the largern-channel MOSFET, there is a significant increase in current drivecapability, frequency response and transconductance of the switch, whichin turn results in a significant reduction in settling time of the bus175.

While the example above describes asymmetric switches 191 for the outputdrivers 198 ₁ and 198 ₂ in which the n-channel MOSFET is larger than thep-channel MOSFET, it should be appreciated that in another embodiment,the converse may be implemented, namely, asymmetric switches in whichthe p-channel MOSFET is larger than the n-channel MOSFET. In one aspectof this embodiment, with reference again to FIG. 16, the current sink197 may alternatively serve as a source of current to appropriatelydrive the output of the column output buffer (see the buffer amplifier111 j of FIG. 9) of a selected column, and be configured such that thebus 175 is normally “pulled up” when all switches 191 ₂, 191 ₄, . . .191 ₅₁₂ are open or off (not conducting). In this situation, most of theswitch conduction may be accomplished by the p-channel MOSFET of theCMOS-pair constituting the switch. Benefits of reduced switchcapacitance (and hence reduced bus capacitance) may be realized in thisembodiment, although the overall beneficial effect of reduced settlingtime for the bus 175 may be somewhat less than that described previouslyabove, due to the lower frequency response of p-channel MOSFETs ascompared to n-channel MOSFETs. Nevertheless, asymmetric switches basedon larger p-channel MOSFETs may still facilitate a notable reduction inbus settling time, and may also provide for circuit implementations inwhich the column output buffer amplifier (111 j of FIG. 9) may be abody-tied source follower with appreciably increased gain.

In yet another embodiment directed to facilitating rapid settling of thebus 175 shown in FIG. 16, it may be appreciated that fewer switches 191coupled to the bus 175 results in a smaller bus capacitance. With thisin mind, and with reference again to FIG. 13, in yet another embodiment,more than two output drivers 198 ₁ and 198 ₂ may be employed in theISFET array 100 such that each output driver handles a smaller number ofcolumns of the array. For example, rather than having all even columnshandled by one driver and all odd columns handled by another driver, thearray may include four column select registers 194 _(1.2.3.4) and fourcorresponding output drivers 198 _(1.2.3.4) such that each output driverhandles one-fourth of the total columns of the array, rather thanone-half of the columns. In such an implementation, each output driverwould accordingly have half the number of switches 191 as compared withthe embodiment discussed above in connection with FIG. 16, and the bus175 of each output driver would have a corresponding lower capacitance,thereby improving bus settling time. While four output drivers arediscussed for purposes of illustration in this example, it should beappreciated that the present disclosure is not limited in this respect,and virtually any number of output drivers greater than two may beemployed to improve bus settling time in the scenario described above.Other array embodiments in which more than two output drivers areemployed to facilitate rapid data acquisition from the array arediscussed in greater detail below (e.g., in connection with FIGS.19-23).

For purposes of illustration, the bus 175 may have a capacitance in therange of approximately 5 pF to 20 pF in any of the embodiments discussedimmediately above (e.g. symmetric switches, asymmetric switches, greaternumbers of output drivers, etc.). Of course, it should be appreciatedthat the capacitance of the bus 175 is not limited to these exemplaryvalues, and that other capacitance values are possible in differentimplementations of an array according to the present disclosure.

In one aspect of the array design discussed above in connection withFIGS. 13-16, separate analog supply voltage connections (for VDDA,VSSA), digital supply voltage connections (for VDDD, VSSD) and outputbuffer supply voltage connections (for VDDO, VSSO) are provided on thearray to facilitate noise isolation and reduce signal cross-talk amongstvarious array components, thereby increasing the signal-to-noise ratio(SNR) of the output signals Vout1 and Vout2. In one exemplaryimplementation, the positive supply voltages VDDA, VDDD and VDDO eachmay be approximately 3.3 Volts. In another aspect, these voltagesrespectively may be provided “off chip” by one or more programmablevoltage sources, as discussed further below in connection with FIG. 17.

FIG. 17 illustrates a block diagram of the sensor array 100 of FIG. 13coupled to an array controller 250, according to one inventiveembodiment of the present disclosure. In various exemplaryimplementations, the array controller 250 may be fabricated as a “standalone” controller, or as one or more computer compatible “cards” formingpart of a computer 260, as discussed above in connection with FIG. 8. Inone aspect, the functions of the array controller 250 may be controlledby the computer 260 through an interface block 252 (e.g., serialinterface, via USB port or PCI bus, Ethernet connection, etc.), as shownin FIG. 17. In one embodiment, all or a portion of the array controller250 is fabricated as one or more printed circuit boards, and the array100 is configured to plug into one of the printed circuit boards,similar to a conventional IC chip (e.g., the array 100 is configured asan ASIC that plugs into a chip socket, such as a zero-insertion-force or“ZIF” socket, of a printed circuit board). In one aspect of such anembodiment, an array 100 configured as an ASIC may include one or morepins/terminal connections dedicated to providing an identification code,indicated as “ID” in FIG. 17, that may be accessed/read by the arraycontroller 250 and/or passed on to the computer 260. Such anidentification code may represent various attributes of the array 100(e.g., size, number of pixels, number of output signals, variousoperating parameters such as supply and/or bias voltages, etc.) and maybe processed to determine corresponding operating modes, parameters andor signals provided by the array controller 250 to ensure appropriateoperation with any of a number of different types of arrays 100. In oneexemplary implementation, an array 100 configured as an ASIC may beprovided with three pins dedicated to an identification code, and duringthe manufacturing process the ASIC may be encoded to provide one ofthree possible voltage states at each of these three pins (i.e., atri-state pin coding scheme) to be read by the array controller 250,thereby providing for 27 unique array identification codes. In anotheraspect of this embodiment, all or portions of the array controller 250may be implemented as a field programmable gate array (FPGA) configuredto perform various array controller functions described in furtherdetail below.

Generally, the array controller 250 provides various supply voltages andbias voltages to the array 100, as well as various signals relating torow and column selection, sampling of pixel outputs and dataacquisition. In particular, the array controller 250 reads one or moreanalog output signals (e.g., Vout1 and Vout2) including multiplexedrespective pixel voltage signals from the array 100 and then digitizesthese respective pixel signals to provide measurement data to thecomputer 260, which in turn may store and/or process the data. In someimplementations, the array controller 250 also may be configured toperform or facilitate various array calibration and diagnosticfunctions, and an optional array UV irradiation treatment as discussedabove in connection with FIG. 11A.

As illustrated in FIG. 17, the array controller 250 generally providesto the array 100 the analog supply voltage and ground (VDDA, VSSA), thedigital supply voltage and ground (VDDD, VSSD), and the buffer outputsupply voltage and ground (VDDO, VSSO). In one exemplary implementation,each of the supply voltages VDDA, VDDD and VDDO is approximately 3.3Volts. In another implementation, the supply voltages VDDA, VDDD andVDDO may be as low as approximately 1.8 Volts. As discussed above, inone aspect each of these power supply voltages is provided to the array100 via separate conducting paths to facilitate noise isolation. Inanother aspect, these supply voltages may originate from respectivepower supplies/regulators, or one or more of these supply voltages mayoriginate from a common source in a power supply 258 of the arraycontroller 250. The power supply 258 also may provide the various biasvoltages required for array operation (e.g., VB1, VB2, VB3, VB4, VBO0,V_(BODY)) and the reference voltage VREF used for array diagnostics andcalibration.

In another aspect, the power supply 258 includes one or moredigital-to-analog converters (DACs) that may be controlled by thecomputer 260 to allow any or all of the bias voltages, referencevoltage, and supply voltages to be changed under software control (i.e.,programmable bias settings). For example, a power supply 258 responsiveto computer control (e.g., via software execution) may facilitateadjustment of one or more of the supply voltages (e.g., switchingbetween 3.3 Volts and 1.8 Volts depending on chip type as represented byan identification code), and or adjustment of one or more of the biasvoltages VB1 and VB2 for pixel drain current, VB3 for column bus drive,VB4 for column amplifier bandwidth, and VBO0 for column output buffercurrent drive. In some aspects, one or more bias voltages may beadjusted to optimize settling times of signals from enabled pixels.Additionally, the common body voltage V_(BODY) for all ISFETs of thearray may be grounded during an optional post-fabrication UV irradiationtreatment to reduce trapped charge, and then coupled to a higher voltage(e.g., VDDA) during diagnostic analysis, calibration, and normaloperation of the array for measurement/data acquisition. Likewise, thereference voltage VREF may be varied to facilitate a variety ofdiagnostic and calibration functions.

As also shown in FIG. 17, the reference electrode 76 which is typicallyemployed in connection with an analyte solution to be measured by thearray 100 (as discussed above in connection with FIG. 1), may be coupledto the power supply 258 to provide a reference potential for the pixeloutput voltages. For example, in one implementation the referenceelectrode 76 may be coupled to a supply ground (e.g., the analog groundVSSA) to provide a reference for the pixel output voltages based on Eq.(3) above. In other exemplary implementations, the reference electrodevoltage may be set by placing a solution/sample of interest having aknown pH level in proximity to the sensor array 100 and adjusting thereference electrode voltage until the array output signals Vout1 andVout2 provide pixel voltages at a desired reference level, from whichsubsequent changes in pixel voltages reflect local changes in pH withrespect to the known reference pH level. In general, it should beappreciated that a voltage associated with the reference electrode 76need not necessarily be identical to the reference voltage VREFdiscussed above (which may be employed for a variety of array diagnosticand calibration functions), although in some implementations thereference voltage VREF provided by the power supply 258 may be used toset the voltage of the reference electrode 76.

Regarding data acquisition from the array 100, in one embodiment thearray controller 250 of FIG. 17 may include one or more preamplifiers253 to further buffer one or more output signals (e.g., Vout1 and Vout2)from the sensor array and provide selectable gain. In one aspect, thearray controller 250 may include one preamplifier for each output signal(e.g., two preamplifiers for two analog output signals). In otheraspects, the preamplifiers may be configured to accept input voltagesfrom 0.0 to 1.8 Volts or 0.0 to 3.3 Volts, may haveprogrammable/computer selectable gains (e.g., 1, 2, 5, 10 and 20) andlow noise outputs (e.g., <10 nV/sqrtHz), and may provide low passfiltering (e.g., bandwidths of 5 MHz and 25 MHz). With respect to noisereduction and increasing signal-to-noise ratio, in one implementation inwhich the array 100 is configured as an ASIC placed in a chip socket ofa printed circuit board containing all or a portion of the arraycontroller 250, filtering capacitors may be employed in proximity to thechip socket (e.g., the underside of a ZIF socket) to facilitate noisereduction. In yet another aspect, the preamplifiers may have aprogrammable/computer selectable offset for input and/or output voltagesignals to set a nominal level for either to a desired range.

The array controller 250 of FIG. 17 also comprises one or moreanalog-to-digital converters 254 (ADCs) to convert the sensor arrayoutput signals Vout1 and Vout2 to digital outputs (e.g., 10-bit or12-bit) so as to provide data to the computer 260. In one aspect, oneADC may be employed for each analog output of the sensor array, and eachADC may be coupled to the output of a corresponding preamplifier (ifpreamplifiers are employed in a given implementation). In anotheraspect, the ADC(s) may have a computer-selectable input range (e.g., 50mV, 200 mV, 500 mV, IV) to facilitate compatibility with differentranges of array output signals and/or preamplifier parameters. In yetother aspects, the bandwidth of the ADC(s) may be greater than 60 MHz,and the data acquisition/conversion rate greater than 25 MHz (e.g., ashigh as 100 MHz or greater).

In the embodiment of FIG. 17, ADC acquisition timing and array row andcolumn selection may be controlled by a timing generator 256. Inparticular, the timing generator provides the digital vertical data andclock signals (DV, CV) to control row selection, the digital horizontaldata and clock signals (DH, CH) to control column selection, and thecolumn sample and hold signal COL SH to sample respective pixel voltagesfor an enabled row, as discussed above in connection with FIG. 9. Thetiming generator 256 also provides a sampling clock signal CS to theADC(s) 254 so as to appropriately sample and digitize consecutive pixelvalues in the data stream of a given array analog output signal (e.g.,Vout1 and Vout2), as discussed further below in connection with FIG. 18.In some implementations, the timing generator 256 may be implemented bya microprocessor executing code and configured as a multi-channeldigital pattern generator to provide appropriately timed controlsignals. In one exemplary implementation, the timing generator 256 maybe implemented as a field-programmable gate array (FPGA).

FIG. 18 illustrates an exemplary timing diagram for various arraycontrol signals, as provided by the timing generator 256, to acquirepixel data from the sensor array 100. For purposes of the followingdiscussion, a “frame” is defined as a data set that includes a value(e.g., pixel output signal or voltage V_(S)) for each pixel in thearray, and a “frame rate” is defined as the rate at which successiveframes may be acquired from the array. Thus, the frame rate correspondsessentially to a “pixel sampling rate” for each pixel of the array, asdata from any given pixel is obtained at the frame rate.

In the example of FIG. 18, an exemplary frame rate of 20 frames/sec ischosen to illustrate operation of the array (i.e., row and columnselection and signal acquisition); however, it should be appreciatedthat arrays and array controllers according to the present disclosureare not limited in this respect, as different frame rates, includinglower frame rates (e.g., 1 to 10 frames/second) or higher frame rates(e.g., 25, 30, 40, 50, 60, 70 to 100 frames/sec., etc.), with arrayshaving the same or higher numbers of pixels, are possible. In someexemplary applications, a data set may be acquired that includes manyframes over several seconds to conduct an experiment on a given analyteor analytes. Several such experiments may be performed in succession, insome cases with pauses in between to allow for data transfer/processingand/or washing of the sensor array ASIC and reagent preparation for asubsequent experiment.

For example, with respect to the method for detecting nucleotideincorporation, appropriate frame rates may be chosen to sufficientlysample the ISFET's output signal. In some exemplary implementations, ahydrogen ion signal may have a full-width at half-maximum (FWHM) on theorder of approximately 1 second to approximately 2.5 seconds, dependingon the number of nucleotide incorporation events. Given these exemplaryvalues, a frame rate (or pixel sampling rate) of 20 Hz is sufficient toreliably resolve the signals in a given pixel's output signal. Again,the frame rates given in this example are provided primarily forpurposes of illustration, and different frame rates may be involved inother implementations.

In one implementation, the array controller 250 controls the array 100to enable rows successively, one at a time. For example, with referenceagain for the moment to FIG. 9, a first row of pixels is enabled via therow select signal RowSel₁ . The enabled pixels are allowed to settle forsome time period, after which the COL SH signal is asserted briefly toclose the sample/hold switch in each column and store on the column'ssample/hold capacitor C_(sh) the voltage value output by the first pixelin the column. This voltage is then available as the column outputvoltage V_(COLj) applied to one of the two (odd and even column) arrayoutput drivers 198 ₁ and 198 ₂ (e.g., see FIG. 16). The COL SH signal isthen de-asserted, thereby opening the sample/hold switches in eachcolumn and decoupling the column output buffer 111 j from the columnamplifiers 107A and 107B. Shortly thereafter, the second row of pixelsis enabled via the row select signal RowSel₂ . During the time period inwhich the second row of pixels is allowed to settle, the column selectsignals are generated two at a time (one odd and one even; odd columnselect signals are applied in succession to the odd output driver, evencolumn select signals are applied in succession to the even outputdriver) to read the column output voltages associated with the firstrow. Thus, while a given row in the array is enabled and settling, theprevious row is being read out, two columns at a time. By staggering rowselection and sampling/readout (e.g., via different vertical andhorizontal clock signals and column sample/hold), and by readingmultiple columns at a time for a given row, a frame of data may beacquired from the array in a significantly streamlined manner.

FIG. 18 illustrates the timing details of the foregoing process for anexemplary frame rate of 20 frames/sec. Given this frame rate and 512rows in the array, each row must be read out in approximately 98microseconds, as indicated by the vertical delineations in FIG. 18.Accordingly, the vertical clock signal CV has a period of 98microseconds (i.e., a clock frequency of over 10 kHz), with a new rowbeing enabled on a trailing edge (negative transition) of the CV signal.The left side of FIG. 18 reflects the beginning of a new frame cycle, atwhich point the vertical data signal DV is asserted before a firsttrailing edge of the CV signal and de-asserted before the next trailingedge of the CV signal (for data acquisition from successive frames, thevertical data signal is reasserted again only after row 512 is enabled).Also, immediately before each trailing edge of the CV signal (i.e., newrow enabled), the COL SH signal is asserted for 2 microseconds, leavingapproximately 50 nanoseconds before the trailing edge of the CV signal.

In FIG. 18, the first occurrence of the COL SH signal is actuallysampling the pixel values of row 512 of the array. Thus, upon the firsttrailing edge of the CV signal, the first row is enabled and allowed tosettle (for approximately 96 microseconds) until the second occurrenceof the COL SH signal. During this settling time for the first row, thepixel values of row 512 are read out via the column select signals.Because two column select signals are generated simultaneously to read512 columns, the horizontal clock signal CH must generate 256 cycleswithin this period, each trailing edge of the CH signal generating oneodd and one even column select signal. As shown in FIG. 18, the firsttrailing edge of the CH signal in a given row is timed to occur twomicroseconds after the selection of the row (after deactivation of theCOL SH signal) to allow for settling of the voltage values stored on thesample/hold capacitors C_(sh) and provided by the column output buffers.It should be appreciated however that, in other implementations (e.g.,as discussed below in connection with FIG. 18A), the time period betweenthe first trailing edge of the CH signal and a trailing edge (i.e.,deactivation) of the COL SH signal may be significantly less than twomicroseconds, and in some cases as small as just over 50 nanoseconds.Also for each row, the horizontal data signal DH is asserted before thefirst trailing edge of the CH signal and de-asserted before the nexttrailing edge of the CH signal. The last two columns (e.g., 511 and 512)are selected before the occurrence of the COL SH signal which, asdiscussed above, occurs approximately two microseconds before the nextrow is enabled. Thus, 512 columns are read, two at a time, within a timeperiod of approximately 94 microseconds (i.e., 98 microseconds per row,minus two microseconds at the beginning and end of each row). Thisresults in a data rate for each of the array output signals Vout1 andVout2 of approximately 2.7 MHz.

FIG. 18A illustrates another timing diagram of a data acquisitionprocess from an array 100 that is slightly modified from the timingdiagram of FIG. 18. As discussed above in connection with FIG. 13, insome implementations an array similar to that shown in FIG. 13 may beconfigured to include a region of 512 by 512 “active” pixels that aresurrounded by a perimeter of reference pixels (i.e., the first and lasttwo rows and columns of the array), resulting in an array having a totalpixel count of 516 by 516 pixels. Accordingly, given the exemplary framerate of 20 frames/sec and 516 rows in the array, each row must be readout in approximately 97 microseconds, as indicated by the verticaldelineations in FIG. 18A. Accordingly, the vertical clock signal CV hasa slightly smaller period of 97 microseconds. Because two column selectsignals are generated simultaneously to read 516 columns, the horizontalclock signal CH must generate 258 cycles within this period, as opposedto the 256 cycles referenced in connection with FIG. 18. Accordingly, inone aspect illustrated in FIG. 18A, the first trailing edge of the CHsignal in a given row is timed to occur just over 50 nanoseconds fromthe trailing edge (i.e., deactivation) of the COL SH signal, so as to“squeeze” additional horizontal clock cycles into a slightly smallerperiod of the vertical clock signal CV. As in FIG. 18, the horizontaldata signal DH is asserted before the first trailing edge of the CHsignal, and as such also occurs slightly earlier in the timing diagramof FIG. 18A as compared to FIG. 18. The last two columns (i.e., columns515 and 516, labeled as “Ref3,4 in FIG. 18A) are selected before theoccurrence of the COL SH signal which, as discussed above, occursapproximately two microseconds before the next row is enabled. Thus, 516columns are read, two at a time, within a time period of approximately95 microseconds (i.e., 97 microseconds per row, minus two microsecondsat the end of each row and negligible time at the beginning of eachrow). This results in essentially the same data rate for each of thearray output signals Vout1 and Vout2 provided by the timing diagram ofFIG. 18, namely, approximately 2.7 MHz.

As discussed above in connection with FIG. 17, the timing generator 256also generates the sampling clock signal CS to the ADC(s) 254 so as toappropriately sample and digitize consecutive pixel values in the datastream of a given array output signal. In one aspect, the sampling clocksignal CS provides for sampling a given pixel value in the data streamat least once. Although the sampling clock signal CS is not shown in thetiming diagrams of FIGS. 18 and 18A, it may be appreciated that inexemplary implementations the signal CS may essentially track the timingof the horizontal clock signal CH; in particular, the sampling clocksignal CS may be coordinated with the horizontal clock signal CH so asto cause the ADC(s) to sample a pixel value immediately prior to a nextpixel value in the data stream being enabled by CH, thereby allowing foras much signal settling time as possible prior to sampling a given pixelvalue. For example, the ADC(s) may be configured to sample an inputpixel value upon a positive transition of CS, and respective positivetransitions of CS may be timed by the timing generator 256 to occurimmediately prior to, or in some cases essentially coincident with,respective negative transitions of CH, so as to sample a given pixeljust prior to the next pixel in the data stream being enabled. Inanother exemplary implementation, the ADC(s) 254 may be controlled bythe timing generator 256 via the sampling clock signal CS to sample theoutput signals Vout1 and Vout2 at a significantly higher rate to providemultiple digitized samples for each pixel measurement, which may then beaveraged (e.g., the ADC data acquisition rate may be approximately 100MHz to sample the 2.7 MHz array output signals, thereby providing asmany as approximately 35-40 samples per pixel measurement).

In one embodiment, once pixel values are sampled and digitized by theADC(s) 254, the computer 260 may be programmed to process pixel dataobtained from the array 100 and the array controller 250 so as tofacilitate high data acquisition rates that in some cases may exceed asufficient settling time for pixel voltages represented in a given arrayoutput signal. A flow chart illustrating an exemplary method accordingto one embodiment of the present invention that may be implemented bythe computer 260 for processing and correction of array data acquired athigh acquisition rates is illustrated in FIG. 18B. In various aspects ofthis embodiment, the computer 260 is programmed to first characterize asufficient settling time for pixel voltages in a given array outputsignal, as well as array response at appreciably high operatingfrequencies, using a reference or “dry” input to the array (e.g., noanalyte present). This characterization forms the basis for derivingcorrection factors that are subsequently applied to data obtained fromthe array at the high operating frequencies and in the presence of ananalyte to be measured.

Regarding pixel settling time, with reference again to FIG. 16, asdiscussed above a given array output signal (e.g., Vout2 in FIG. 16)includes a series of pixel voltage values resulting from the sequentialoperation of the column select switches 191 to apply respective columnvoltages V_(COLj) via the bus 175 to the buffer amplifier 199 (therespective column voltages V_(COLj) in turn represent buffered versionsof ISFET source voltages V_(Sj)). In some implementations, it isobserved that voltage changes ΔV_(PIX) in the array output signalbetween two consecutive pixel reads is characterized as an exponentialprocess given by

ΔV _(PIX)(t)=A(1−e ^(−1/k))  (PP)

where A is the difference (V_(COLj)-V_(COLj-1)) between two pixelvoltage values and k is a time constant associated with a capacitance ofthe bus 175. FIGS. 18C and 18D illustrate exemplary pixel voltages in agiven array output signal Vout (e.g., one of Vout1 and Vout2) showingpixel-to-pixel transitions in the output signal as a function of time,plotted against exemplary sampling clock signals CS. In FIG. 18C, thesampling clock signal CS has a period 524, and an ADC controlled by CSsamples a pixel voltage upon a positive transition of CS (as discussedabove, in one implementation CS and CH have essentially a same period).FIG. 18C indicates two samples 525A and 525B, between which anexponential voltage transition 522 corresponding to ΔV_(PIX) (t),between a voltage difference A, may be readily observed.

For purposes of the present discussion, pixel “settling time” t_(settle)is defined as the time t at which ΔV_(PIX)(t) attains a value thatdiffers from it's final value by an amount that is equal to the peaknoise level of the array output signal. If the peak noise level of thearray output signal is denoted as n_(p), then the voltage at thesettling time t_(settle) is given byΔV_(PIX)(t_(settle))=A[1−(n_(p)/A)]. Substituting in Eq. (PP) andsolving for t_(settle) yields

$\begin{matrix}{t_{settle} = {{- k}\; {{\ln \left( \frac{n_{p}}{A} \right)}.}}} & ({QQ})\end{matrix}$

FIG. 18D conceptually illustrates a pixel settling time t_(settle)(reference numeral 526) for a single voltage transition 522 between twopixel voltages having a difference A, using a sampling clock signal CShaving a sufficiently long period so as to allow for full settling. Toprovide some exemplary parameters for purposes of illustration, in oneimplementation a maximum value for A, representing a maximum range forpixel voltage transitions (e.g., consecutive pixels at minimum andmaximum values), is on the order of approximately 250 mV. Additionally,a peak noise level n_(p) of the array output signal is taken asapproximately 100 μV, and the time constant k is taken as 5 nanoseconds.These values provide an exemplary settling time t_(settle) ofapproximately 40 nanoseconds. If a maximum data rate of an array outputsignal is taken as the inverse of the settling time t_(settle), asettling time of 40 nanoseconds corresponds to maximum data rate of 25MHz. In other implementations, A may be on the order of 20 mV and thetime constant k may be on the order of 15 nanoseconds, resulting in asettling time t_(settle) of approximately 80 nanoseconds and a maximumdata rate of 12.5 MHz. The values of k indicated above generallycorrespond to capacitances for the bus 175 in a range of approximately 5pF to 20 pF. It should be appreciated that the foregoing values areprovided primarily for purposes of illustration, and that variousembodiments of the present invention are not limited to these exemplaryvalues; in particular, arrays according to various embodiment of thepresent invention may have different pixel settling times t_(settle)(e.g., in some cases less than 40 nanoseconds).

As indicated above, in one embodiment pixel data may be acquired fromthe array at data rates that exceed those dictated by the pixel settlingtime. FIG. 18B illustrates a flow chart for such a method according toone inventive embodiment of the present disclosure. In the method ofFIG. 18B, sufficiently slow clock frequencies initially are chosen forthe signals CV, CH and CS such that the resulting data rate per arrayoutput signal is equal to or lower than the reciprocal of the pixelsettling time t_(settle) to allow for fully settled pixel voltage valuesfrom pixel to pixel in a given output signal. With these clockfrequencies, as indicated in block 502 of FIG. 18B, settled pixelvoltage values are then acquired for the entire array in the absence ofan analyte (or in the presence of a reference analyte) to provide afirst “dry” or reference data image for the array. In block 504 of FIG.18B, for each pixel voltage constituting the first data image, atransition value between the pixel's final voltage and the final voltageof the immediately preceding pixel in the corresponding output signaldata stream (i.e., the voltage difference A) is collected and stored.The collection of these transition values for all pixels of the arrayprovides a first transition value data set.

Subsequently, in block 506 of FIG. 18B, the clock frequencies for thesignals CV, CH and CS are increased such that the resulting data rateper array output signal exceeds a rate at which pixel voltage values arefully settled (i.e., a data rate higher than the reciprocal of thesettling time t_(settle)). For purposes of the present discussion, thedata rate per array output signal resulting from the selection of suchincreased clock frequencies for the signals CV, CH and CS is referred toas an “overspeed data rate.” Using the clock frequencies correspondingto the overspeed data rate, pixel voltage values are again obtained forthe entire array in the absence of an analyte (or in the presence of thesame reference analyte) to provide a second “dry” or reference dataimage for the array. In block 508 of FIG. 18B, a second transition valuedata set based on the second data image obtained at the overspeed datarate is calculated and stored, as described above for the first dataimage.

In block 510 of FIG. 18B, a correction factor for each pixel of thearray is calculated based on the values stored in the first and secondtransition value data sets. For example, a correction factor for eachpixel may be calculated as a ratio of its transition value from thefirst transition value data set and its corresponding transition valuefrom the second transition value data set (e.g., the transition valuefrom the first data set may be divided by the transition value from thesecond data set, or vice versa) to provide a correction factor data setwhich is then stored. As noted in blocks 512 and 514 of FIG. 18B, thiscorrection factor data set may then be employed to process pixel dataobtained from the array operated at clock frequencies corresponding tothe overspeed data rate, in the presence of an actual analyte to bemeasured; in particular, data obtained from the array at the overspeeddata rate in the presence of an analyte may be multiplied or divided asappropriate by the correction factor data set (e.g., each pixelmultiplied or divided by a corresponding correction factor) to obtaincorrected data representative of the desired analyte property to bemeasured (e.g., ion concentration). It should be appreciated that oncethe correction factor data set is calculated and stored, it may be usedrepeatedly to correct multiple frames of data acquired from the array atthe overspeed data rate.

In addition to controlling the sensor array and ADCs, the timinggenerator 256 may be configured to facilitate various array calibrationand diagnostic functions, as well as an optional UV irradiationtreatment. To this end, the timing generator may utilize the signal LSTVindicating the selection of the last row of the array and the signalLSTH to indicate the selection of the last column of the array. Thetiming generator 256 also may be responsible for generating the CALsignal which applies the reference voltage VREF to the column bufferamplifiers, and generating the UV signal which grounds the drains of allISFETs in the array during a UV irradiation process (see FIG. 9). Thetiming generator also may provide some control function over the powersupply 258 during various calibration and diagnostic functions, or UVirradiation, to appropriately control supply or bias voltages; forexample, during UV irradiation, the timing generator may control thepower supply to couple the body voltage V_(BODY) to ground while the UVsignal is activated to ground the ISFET drains. With respect to arraycalibration and diagnostics, as well as UV irradiation, in someimplementations the timing generator may receive specialized programsfrom the computer 260 to provide appropriate control signals. In oneaspect, the computer 260 may use various data obtained from referenceand/or dummy pixels of the array, as well as column information based onthe application of the CAL signal and the reference voltage VREF, todetermine various calibration parameters associated with a given arrayand/or generate specialized programs for calibration and diagnosticfunctions.

Having discussed several aspects of an exemplary ISFET array, FIGS.19-23 illustrate block diagrams of alternative CMOS IC chipimplementations of ISFET sensor arrays having greater numbers of pixels,according to yet other inventive embodiments. In one aspect, each of theISFET arrays discussed further below in connection with FIGS. 19-23 maybe controlled by an array controller similar to that shown in FIG. 17,in some cases with minor modifications to accommodate higher numbers ofpixels (e.g., additional preamplifiers 253 and analog-to-digitalconverters 254).

FIG. 19 illustrates a block diagram of an ISFET sensor array 100A basedon the column and pixel designs discussed above in connection with FIGS.9-12 and a 0.35 micrometer CMOS fabrication process, according to oneinventive embodiment. The array 100A includes 2048 columns 102 ₁ through102 ₂₀₄₈, wherein each column includes 2048 geometrically square pixels105 ₁ through 105 ₂₀₄₈, each having a size of approximately 9micrometers by 9 micrometers. Thus, the array includes over four millionpixels (>4 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 20.5millimeters by 20.5 millimeters.

In one aspect of the embodiment shown in FIG. 19, the array 100A may beconfigured, at least in part, as multiple groups of pixels that may berespectively controlled. For example, each column of pixels may bedivided into top and bottom halves, and the collection of pixels inrespective top halves of columns form a first group 400 ₁ of rows (e.g.,a top group, rows 1-1024) and the collection of pixels in respectivebottom halves of columns form a second group 400 ₂ of rows (e.g., abottom group, rows 1025-2048). In turn; each of the first and second(e.g., top and bottom) groups of rows is associated with correspondingrow select registers, column bias/readout circuitry, column selectregisters, and output drivers. In this manner, pixel selection and dataacquisition from each of the first and second groups of rows 400 ₁ and400 ₂ is substantially similar to pixel selection and data acquisitionfrom the entire array 100 shown in FIG. 13; stated differently, in oneaspect, the array 100A of FIG. 19 substantially comprises twosimultaneously controlled “sub-arrays” of different pixel groups toprovide for significantly streamlined data acquisition from highernumbers of pixels.

In particular, FIG. 19 shows that row selection of the first group 400 ₁of rows may be controlled by a first row select register 192 ₁, and rowselection of the second group 400 ₂ of rows may be controlled by asecond row select register 192 ₂. In one aspect, each of the row selectregisters 192 ₁ and 192 ₂ may be configured as discussed above inconnection with FIG. 14 to receive vertical clock (CV) and vertical data(DV) signals and generate row select signals in response; for examplethe first row select register 192 ₁ may generate the signals RowSel₁through RowSel₁₀₂₄ and the second row select register 192 ₂ may generatethe signals RowSel₁₀₂₅ through RowSel₂₀₄₈ . In another aspect, both rowselect registers 192 ₁ and 192 ₂ may simultaneously receive commonvertical clock and data signals, such that two rows of the array areenabled at any given time, one from the top group and another from thebottom group.

For each of the first and second groups of rows, the array 100A of FIG.19 further comprises column bias/readout circuitry 110 _(1T)-110_(2048T) (for the first row group 400 ₁) and 110 _(1B)-110 _(2048B) (forthe second row group 400 ₂), such that each column includes twoinstances of the bias/readout circuitry 110 j shown in FIG. 9. The array100A also comprises two column select registers 192 _(1,2) (odd andeven) and two output drivers 198 _(1,2) (odd and even) for the secondrow group 400 ₂, and two column select registers 192 _(3,4) (odd andeven) and two output drivers 198 _(3,4) (odd and even) for the first rowgroup 400 ₁ (i.e., a total of four column select registers and fouroutput drivers). The column select registers receive horizontal clocksignals (CHT and CHB for the first row group and second row group,respectively) and horizontal data signals (DHT and DHB for the first rowgroup and second row group, respectively) to control odd and even columnselection. In one implementation, the CHT and CHB signals may beprovided as common signals, and the DHT and DHB may be provided ascommon signals, to simultaneously read out four columns at a time fromthe array (i.e., one odd and one even column from each row group); inparticular, as discussed above in connection with FIGS. 13-18, twocolumns may be simultaneously read for each enabled row and thecorresponding pixel voltages provided as two output signals. Thus, viathe enablement of two rows at any given time, and reading of two columnsper row at any given time, the array 100A may provide four simultaneousoutput signals Vout1, Vout2, Vout3 and Vout4.

In one exemplary implementation of the array 100A of FIG. 19, in whichcomplete data frames (all pixels from both the first and second rowgroups 400 ₁ and 400 ₂) are acquired at a frame rate of 20 frames/sec,1024 pairs of rows are successively enabled for periods of approximately49 microseconds each. For each enabled row, 1024 pixels are read out byeach column select register/output driver during approximately 45microseconds (allowing 2 microseconds at the beginning and end of eachrow, as discussed above in connection with FIG. 18). Thus, in thisexample, each of the array output signals Vout1, Vout2, Vout3 and Vout4has a data rate of approximately 23 MHz. Again, it should be appreciatedthat in other implementations, data may be acquired from the array 100Aof FIG. 19 at frame rates other than 20 frames/sec (e.g., 50-100frames/sec).

Like the array 100 of FIG. 13, in yet other aspects the array 100A ofFIG. 19 may include multiple rows and columns of dummy or referencepixels 103 around a perimeter of the array to facilitate preliminarytest/evaluation data, offset determination an/or array calibration.Additionally, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array 100A in block 195, in a manner similar to that discussed abovein connection with FIG. 13.

FIG. 20 illustrates a block diagram of an ISFET sensor array 100B basedon a 0.35 micrometer CMOS fabrication process and having a configurationsubstantially similar to the array 100A discussed above in FIG. 19,according to yet another inventive embodiment. While the array 100B alsois based generally on the column and pixel designs discussed above inconnection with FIGS. 9-12, the pixel size/pitch in the array 100B issmaller than that of the pixel shown in FIG. 10. In particular, withreference again to FIGS. 10 and 11, the dimension “e” shown in FIG. 10is substantially reduced in the embodiment of FIG. 20, without affectingthe integrity of the active pixel components disposed in the centralregion of the pixel, from approximately 9 micrometers to approximately 5micrometers; similarly, the dimension “f” shown in FIG. 10 is reducedfrom approximately 7 micrometers to approximately 4 micrometers. Stateddifferently, some of the peripheral area of the pixel surrounding theactive components is substantially reduced with respect to thedimensions given in connection with FIG. 10, without disturbing thetop-view and cross-sectional layout and design of the pixel's activecomponents as shown in FIGS. 10 and 11. A top view of such a pixel 105Ais shown in FIG. 20A, in which the dimension “e” is 5.1 micrometers andthe dimension “f” is 4.1 micrometers. In one aspect of this pixeldesign, to facilitate size reduction, fewer body connections B areincluded in the pixel 105A (e.g., one at each corner of the pixel) ascompared to the pixel shown in FIG. 10, which includes several bodyconnections B around the entire perimeter of the pixel.

As noted in FIG. 20, the array 100B includes 1348 columns 102 ₁ through102 ₁₃₄₈, wherein each column includes 1152 geometrically square pixels105A₁ through 105A₁₁₅₂, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 1.5 millionpixels (>1.5 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 9millimeters by 9 millimeters. Like the array 100A of FIG. 19, in oneaspect the array 100B of FIG. 20 is divided into two groups of rows 400₁ and 400 ₂, as discussed above in connection with FIG. 19. In oneexemplary implementation, complete data frames (all pixels from both thefirst and second row groups 400 ₁ and 400 ₂) are acquired at a framerate of 50 frames/sec, thereby requiring 576 pairs of rows to besuccessively enabled for periods of approximately 35 microseconds each.For each enabled row, 674 pixels are read out by each column selectregister/output driver during approximately 31 microseconds (allowing 2microseconds at the beginning and end of each row, as discussed above inconnection with FIG. 18). Thus, in this example, each of the arrayoutput signals Vout1, Vout2, Vout3 and Vout4 has a data rate ofapproximately 22 MHz. Again, it should be appreciated that in otherimplementations, data may be acquired from the array 100B of FIG. 20 atframe rates other than 50 frames/sec.

FIG. 21 illustrates a block diagram of an ISFET sensor array 100C basedon a 0.35 micrometer CMOS fabrication process and incorporating thesmaller pixel size discussed above in connection with FIGS. 20 and 20A(5.1 micrometer square pixels), according to yet another embodiment. Asnoted in FIG. 21, the array 100C includes 4000 columns 102 ₁ through 102₄₀₀₀, wherein each column includes 3600 geometrically square pixels105A₁ through 105A₃₆₀₀, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 14 millionpixels (>14 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 22millimeters by 22 millimeters. Like the arrays 100A and 100B of FIGS. 19and 20, in one aspect the array 100C of FIG. 21 is divided into twogroups of rows 400 ₁ and 400 ₂. However, unlike the arrays 100A and100B, for each row group the array 100C includes sixteen column selectregisters and sixteen output drivers to simultaneously read sixteenpixels at a time in an enabled row, such that thirty-two output signalsVout1-Vout32 may be provided from the array 100C. In one exemplaryimplementation, complete data frames (all pixels from both the first andsecond row groups 400 ₁ and 400 ₂) may be acquired at a frame rate of 50frames/sec, thereby requiring 1800 pairs of rows to be successivelyenabled for periods of approximately 11 microseconds each. For eachenabled row, 250 pixels (4000/16) are read out by each column selectregister/output driver during approximately 7 microseconds (allowing 2microseconds at the beginning and end of each row). Thus, in thisexample, each of the array output signals Vout1-Vout32 has a data rateof approximately 35 MHz. As with the previous embodiments, it should beappreciated that in other implementations, data may be acquired from thearray 100C at frame rates other than 50 frames/sec.

While the exemplary arrays discussed above in connection with FIGS.13-21 are based on a 0.35 micrometer conventional CMOS fabricationprocess, it should be appreciated that arrays according to the presentdisclosure are not limited in this respect, as CMOS fabricationprocesses having feature sizes of less than 0.35 micrometers may beemployed (e.g., 0.18 micrometer CMOS processing techniques) to fabricatesuch arrays. Accordingly, ISFET sensor arrays with a pixel size/pitchsignificantly below 5 micrometers may be fabricated, providing forsignificantly denser ISFET arrays. For example, FIGS. 22 and 23illustrate respective block diagrams of ISFET sensor arrays 100D and100E according to yet other embodiments based on a 0.18 micrometer CMOSfabrication process, in which a pixel size of 2.6 micrometers isachieved. The pixel design itself is based substantially on the pixel105A shown in FIG. 20A, albeit on a smaller scale due to the 0.18micrometer CMOS process.

The array 100D of FIG. 22 includes 2800 columns 102 ₁ through 102 ₂₈₀₀,wherein each column includes 2400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 6.5 million pixels (>6.5 Mega-pixels) and, inone exemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 9 millimeters by 9 millimeters. Likethe arrays 100A, 100B and 100C of FIGS. 19-21, in one aspect the array100D of FIG. 22 is divided into two groups of rows 400 ₁ and 400 ₂.However, unlike the arrays 100A, 100B, and 100C, for each row group thearray 100D includes eight column select registers and eight outputdrivers to simultaneously read eight pixels at a time in an enabled row,such that sixteen output signals Vout1-Vout16 may be provided from thearray 100D. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 50 frames/sec, thereby requiring 1200 pairsof rows to be successively enabled for periods of approximately 16-17microseconds each. For each enabled row, 350 pixels (2800/8) are readout by each column select register/output driver during approximately 14microseconds (allowing 1 to 2 microseconds at the beginning and end ofeach row). Thus, in this example, each of the array output signalsVout1-Vout16 has a data rate of approximately 25 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 50 frames/sec.

The array 100E of FIG. 23 includes 7400 columns 102 ₁ through 102 ₇₄₀₀,wherein each column includes 7400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 54 million pixels (>54 Mega-pixels) and, in oneexemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 21 millimeters by 21 millimeters.Like the arrays 100A-100D of FIGS. 19-22, in one aspect the array 100Eof FIG. 23 is divided into two groups of rows 400 ₁ and 400 ₂. However,unlike the arrays 100A-100D, for each row group the array 100E includesthirty-two column select registers and thirty-two output drivers tosimultaneously read thirty-two pixels at a time in an enabled row, suchthat sixty-four output signals Vout1-Vout64 may be provided from thearray 100E. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 100 frames/sec, thereby requiring 3700 pairsof rows to be successively enabled for periods of approximately 3microseconds each. For each enabled row, 230 pixels (7400/32) are readout by each column select register/output driver during approximately700 nanoseconds. Thus, in this example, each of the array output signalsVout1-Vout64 has a data rate of approximately 328 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 100 frames/sec.

Thus, in various examples of ISFET arrays based on the inventiveconcepts disclosed herein, an array pitch of approximately nine (9)micrometers (e.g., a sensor surface area of less than ten micrometers byten micrometers) allows an ISFET array including over 256,000 pixels(i.e., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (i.e., a 2048 by 2048 array, over 4Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die.In other examples, an array pitch of approximately 5 micrometers allowsan ISFET array including approximately 1.55 Mega-pixels (i.e., a 1348 by1152 array) and associated electronics to be fabricated on a 9millimeter by 9 millimeter die, and an ISFET sensor array including over14 Mega-pixels and associated electronics on a 22 millimeter bymillimeter die. In yet other implementations, using a CMOS fabricationprocess in which feature sizes of less than 0.35 micrometers arepossible (e.g., 0.18 micrometer CMOS processing techniques), ISFETsensor arrays with a pixel size/pitch significantly below 5 micrometersmay be fabricated (e.g., array pitch of 2.6 micrometers or pixel/sensorarea of less than 8 or 9 micrometers²), providing for significantlydense ISFET arrays.

In the embodiments of ISFET arrays discussed above, array pixels employa p-channel ISFET, as discussed above in connection with FIG. 9. Itshould be appreciated, however, that ISFET arrays according to thepresent disclosure are not limited in this respect, and that in otherembodiments pixel designs for ISFET arrays may be based on an n-channelISFET. In particular, any of the arrays discussed above in connectionwith FIGS. 13 and 19-23 may be implemented with pixels based onn-channel ISFETs.

For example, FIG. 24 illustrates the pixel design of FIG. 9 implementedwith an n-channel ISFET and accompanying n-channel MOSFETs, according toanother inventive embodiment of the present disclosure. Morespecifically, FIG. 24 illustrates one exemplary pixel 105 ₁ of an arraycolumn (i.e., the first pixel of the column), together with columnbias/readout circuitry 110 j, in which the ISFET 150 (Q1) is ann-channel ISFET. Like the pixel design of FIG. 9, the pixel design ofFIG. 24 includes only three components, namely, the ISFET 150 and twon-channel MOSFET switches Q2 and Q3, responsive to one of n row selectsignals (RowSel₁ through RowSel_(n), logic high active). No transmissiongates are required in the pixel of FIG. 24, and all devices of the pixelare of a “same type,” i.e., n-channel devices. Also like the pixeldesign of FIG. 9, only four signal lines per pixel, namely the lines 112₁, 114 ₁, 116 ₁ and 118 ₁, are required to operate the three componentsof the pixel 105 ₁ shown in FIG. 24. In other respects, the pixeldesigns of FIG. 9 and FIG. 24 are similar, in that they are bothconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel.

One of the primary differences between the n-channel ISFET pixel designof FIG. 24 and the p-channel ISFET design of FIG. 9 is the oppositedirection of current flow through the pixel. To this end, in FIG. 24,the element 106 _(j) is a controllable current sink coupled to theanalog circuitry supply voltage ground VSSA, and the element 108 _(j) ofthe bias/readout circuitry 110 j is a controllable current sourcecoupled to the analog positive supply voltage VDDA. Additionally, thebody connection of the ISFET 150 is not tied to its source, but ratherto the body connections of other ISFETs of the array, which in turn iscoupled to the analog ground VSSA, as indicated in FIG. 24.

In addition to the pixel designs shown in FIGS. 9 and 24 (based on aconstant ISFET drain current and constant ISFET drain-source voltage),alternative pixel designs are contemplated for ISFET arrays, based onboth p-channel ISFETs and n-channel ISFETs, according to yet otherinventive embodiments of the present disclosure, as illustrated in FIGS.25-27. As discussed below, some alternative pixel designs may requireadditional and/or modified signals from the array controller 250 tofacilitate data acquisition. In particular, a common feature of thepixel designs shown in FIGS. 25-27 includes a sample and hold capacitorwithin each pixel itself, in addition to a sample and hold capacitor foreach column of the array. While the alternative pixel designs of FIGS.25-27 generally include a greater number of components than the pixeldesigns of FIGS. 9 and 24, the feature of a pixel sample and holdcapacitor enables “snapshot” types of arrays, in which all pixels of anarray may be enabled simultaneously to sample a complete frame andacquire signals representing measurements of one or more analytes inproximity to respective ISFETs of the array. In some applications, thismay provide for higher data acquisition speeds and/or improved signalsensitivity (e.g., higher signal-to-noise ratio).

FIG. 25 illustrates one such alternative design for a single pixel 105Cand associated column circuitry 110 j. The pixel 105C employs ann-channel ISFET and is based generally on the premise of providing aconstant voltage across the ISFET Q1 based on a feedback amplifier (Q4,Q5 and Q6). In particular, transistor Q4 constitutes the feedbackamplifier load, and the amplifier current is set by the bias voltage VB1(provided by the array controller). Transistor Q5 is a common gateamplifier and transistor Q6 is a common source amplifier. Again, thepurpose of feedback amplifier is to hold the voltage across the ISFET Q1constant by adjusting the current supplied by transistor Q3. TransistorQ2 limits the maximum current the ISFET Q1 can draw (e.g., so as toprevent damage from overheating a very large array of pixels). Thismaximum current is set by the bias voltage VB2 (also provided by thearray controller). In one aspect of the pixel design shown in FIG. 25,power to the pixel 105C may be turned off by setting the bias voltageVB2 to 0 Volts and the bias voltage VB1 to 3.3 Volts. In this manner,the power supplied to large arrays of such pixels may be modulated(turned on for a short time period and then off by the array controller)to obtain ion concentration measurements while at the same time reducingoverall power consumption of the array. Modulating power to the pixelsalso reduces heat dissipation of the array and potential heating of theanalyte solution, thereby also reducing any potentially deleteriouseffects from sample heating.

In FIG. 25, the output of the feedback amplifier (the gate of transistorQ3) is sampled by MOS switch Q7 and stored on a pixel sample and holdcapacitor Csh within the pixel itself. The switch Q7 is controlled by apixel sample and hold signal pSH (provided to the array chip by thearray controller), which is applied simultaneously to all pixels of thearray so as to simultaneously store the readings of all the pixels ontheir respective sample and hold capacitors. In this manner, arraysbased on the pixel design of FIG. 25 may be considered as “snapshot”arrays, in that a full frame of data is sampled at any given time,rather than sampling successive rows of the array. After each pixelvalue is stored on the corresponding pixel sample and hold capacitorCsh, each pixel 105C (ISFET and feedback amplifier) is free to acquireanother pH reading or it can by turned off to conserve power.

In FIG. 25, the pixel values stored on all of the pixel sample and holdcapacitors Csh are applied to the column circuitry 110 j one row at atime through source follower Q8, which is enabled via the transistor Q9in response to a row select signal (e.g., RowSel1). In particular, aftera row is selected and has settled out, the values stored in the pixelsample and hold capacitors are then in turn stored on the column sampleand hold capacitors Csh2, as enabled by the column sample and holdsignal COL SH, and provided as the column output signal V_(COLj).

FIG. 26 illustrates another alternative design for a single pixel 105Dand associated column circuitry 110 j, according to one embodiment ofthe present disclosure. In this embodiment, the ISFET is shown as ap-channel device. At the start of a data acquisition cycle, CMOSswitches controlled by the signals pSH (pixel sample/hold) and pRST(pixel reset) are closed (these signals are supplied by the arraycontroller). This pulls the source of ISFET (Q1) to the voltage VRST.Subsequently, the switch controlled by the signal pRST is opened, andthe source of ISFET Q1 pulls the pixel sample and hold capacitor Csh toa threshold below the level set by pH. The switch controlled by thesignal pSH is then opened, and the pixel output value is coupled, viaoperation of a switch responsive to the row select signal RowSel1, tothe column circuitry 110 j to provide the column output signal V_(COLj).Like the pixel design in the embodiment illustrated in FIG. 25, arraysbased on the pixel 105D are “snapshot” type arrays in that all pixels ofthe array may be operated simultaneously. In one aspect, this designallows a long simultaneous integration time on all pixels followed by ahigh-speed read out of an entire frame of data.

FIG. 27 illustrates yet another alternative design for a single pixel105E and associated column circuitry 110 j, according to one embodimentof the present disclosure. In this embodiment, again the ISFET is shownas a p-channel device. At the start of a data acquisition cycle, theswitches operated by the control signals p1 and pRST are briefly closed.This clears the value stored on the sampling capacitor Csh and allows acharge to be stored on ISFET (Q1). Subsequently, the switch controlledby the signal pSH is closed, allowing the charge stored on the ISFET Q1to be stored on the pixel sample and hold capacitor Csh. The switchcontrolled by the signal pSH is then opened, and the pixel output valueis coupled, via operation of a switch responsive to the row selectsignal RowSel1, to the column circuitry 110 j to provide the columnoutput signal V_(COLj). Gain may be provided in the pixel 105E via theratio of the ISFET capacitance to the Csh cap, i.e., gain=C_(Q1)/C_(sh),or by enabling the pixel multiple times (i.e., taking multiple samplesof the analyte measurement) and accumulating the ISFET output on thepixel sample and hold capacitor Csh without resetting the capacitor(i.e., gain is a function of the number of accumulations). Like theembodiments of FIGS. 25 and 26, arrays based on the pixel 105D are“snapshot” type arrays in that all pixels of the array may be operatedsimultaneously.

Computer Hardware and Software

With respect to the computer interface 252 of the array controller 250,in one exemplary implementation the interface is configured tofacilitate a data rate of approximately 200 MB/sec to the computer 260,and may include local storage of up to 400 MB or greater. The computer260 is configured to accept data at a rate of 200 MB/sec, and processthe data so as to reconstruct an image of the pixels (e.g., which may bedisplayed in false-color on a monitor). For example, the computer may beconfigured to execute a general-purpose program with routines written inC++ or Visual Basic to manipulate the data and display is as desired.

The systems described herein, when used for sequencing, typicallyinvolve a chemFET array supporting reaction chambers, the chemFETs beingcoupled to an interface capable of executing logic that converts thesignals from the chemFETs into sequencing information.

The sequencing information obtained from the system may be delivered toa handheld computing device, such as a personal digital assistant. Thus,in one embodiment, the invention encompasses logic for displaying acomplete genome of an organism on a handheld computing device. Theinvention also encompasses logic adapted for sending data from a chemFETarray to a handheld computing device. Any of such logic may becomputer-implemented.

Microfluidics and Microwell Arrays

Turning from the sensor discussion, we will now be addressing thecombining of the ISFET array with a microwell array and the attendantfluidics. As most of the drawings of the microwell array structure arepresented only in cross-section or showing that array as only a block ina simplified diagram, FIGS. 28A and 28B are provided to assist thereader in beginning to visualize the resulting apparatus inthree-dimensions. FIG. 28A shows a group of round cylindrical wells 2810arranged in an array, while FIG. 28B shows a group of rectangularcross-section wells 2830 arranged in an array. It will be seen that thewells are separated (isolated) from each other by the material 2840forming the well walls. While it is certainly possible to fabricatewells of other cross-sections, in some embodiments it may not beadvantageous to do so. Such an array of microwells sits over theabove-discussed ISFET array, with one or more ISFETs per well. In thesubsequent drawings, when the microwell array is identified, one maypicture one of these arrays.

Fluidic System: Apparatus and Method for Use with High DensityElectronic Sensor Arrays

For many uses, to complete a system for sensing chemical reactions orchemical agents using the above-explained high density electronicarrays, techniques and apparatus are required for delivery to the arrayelements (called “pixels”) fluids containing chemical or biochemicalcomponents for sensing. In this section, exemplary techniques andmethods will be illustrated, which are useful for such purposes, withdesirable characteristics.

As high speed operation of the system may be desired, it is preferredthat the fluid delivery system, insofar as possible, not limit the speedof operation of the overall system.

Accordingly, needs exist not only for high-speed, high-density arrays ofISFETs or other elements sensitive to ion concentrations or otherchemical attributes, or changes in chemical attributes, but also forrelated mechanisms and techniques for supplying to the array elementsthe samples to be evaluated, in sufficiently small reaction volumes asto substantially advance the speed and quality of detection of thevariable to be sensed.

There are two and sometimes three components or subsystems, and relatedmethods, involved in delivery of the subject chemical samples to thearray elements: (1) macrofluidic system of reagent and wash fluidsupplies and appropriate valving and ancillary apparatus, (2) a flowcell and (3) in many applications, a microwell array. Each of thesesubsystems will be discussed, though in reverse order.

Microwell Array

As discussed elsewhere, for many uses, such as in DNA sequencing, it isdesirable to provide over the array of semiconductor sensors acorresponding array of microwells, each microwell being small enoughpreferably to receive only one DNA-loaded bead, in connection with whichan underlying pixel in the array will provide a corresponding outputsignal.

The use of such a microwell array involves three stages of fabricationand preparation, each of which is discussed separately: (1) creating thearray of microwells to result in a chip having a coat comprising amicrowell array layer; (2) mounting of the coated chip to a fluidicinterface; and in the case of DNA sequencing, (3) loading DNA-loadedbead or beads into the wells. It will be understood, of course, that inother applications, beads may be unnecessary or beads having differentcharacteristics may be employed.

The systems described herein can include an array of microfluidicreaction chambers integrated with a semiconductor comprising an array ofchemFETs. In some embodiments, the invention encompasses such an array.The reaction chambers may, for example, be formed in a glass,dielectric, photodefineable or etchable material. The glass material maybe silicon dioxide.

Preferably, the array comprises at least 100,000 chambers. Preferably,each reaction chamber has a horizontal width and a vertical depth thathas an aspect ratio of about 1:1 or less. Preferably, the pitch betweenthe reaction chambers is no more than about microns.

The above-described array can also be provided in a kit for sequencing.Thus, in some embodiments, the invention encompasses a kit comprising anarray of microfluidic reaction chambers integrated with an array ofchemFETs, and one or more amplification reagents.

In some embodiments, the invention encompasses a sequencing apparatuscomprising a dielectric layer overlying a chemFET, the dielectric layerhaving a recess laterally centered atop the chemFET. Preferably, thedielectric layer is formed of silicon dioxide.

Microwell Array Fabrication

Microwell fabrication may be accomplished in a number of ways. Theactual details of fabrication may require some experimentation and varywith the processing capabilities that are available.

In general, fabrication of a high density array of microwells involvesphoto-lithographically patterning the well array configuration on alayer or layers of material such as photoresist (organic or inorganic),a dielectric, using an etching process. The patterning may be done withthe material on the sensor array or it may be done separately and thentransferred onto the sensor array chip, of some combination of the two.However, techniques other than photolithography are not to be excludedif they provide acceptable results.

One example of a method for forming a microwell array is now discussed,starting with reference to FIG. 29. That figure diagrammatically depictsa top view of one corner (i.e., the lower left corner) of the layout ofa chip showing an array 2910 of the individual ISFET sensors 2912 on theCMOS die 2914. Signal lines 2916 and 2918 are used for addressing thearray and reading its output. Block 2920 represents some of theelectronics for the array, as discussed above, and layer 2922 representsa portion of a wall which becomes part of a microfluidics structure, theflow cell, as more fully explained below; the flow cell is thatstructure which provides a fluid flow over the microwell array or overthe sensor surface directly, if there is no microwell structure. On thesurface of the die, a pattern such as pattern 2922 at the bottom left ofFIG. 29 may be formed during the semiconductor processing to form theISFETs and associated circuitry, for use as alignment marks for locatingthe wells over the sensor pixels when the dielectric has covered thedie's surface.

After the semiconductor structures, as shown, are formed, the microwellstructure is applied to the die. That is, the microwell structure can beformed right on the die or it may be formed separately and then mountedonto the die, either approach being acceptable. To form the microwellstructure on the die, various processes may be used. For example, theentire die may be spin-coated with, for example, a negative photoresistsuch as Microchem's SU-8 2015 or a positive resist/polyimide such as HDMicrosystems HD8820, to the desired height of the microwells. Thedesired height of the wells (e.g., about 4-12 μm in the example of onepixel per well, though not so limited as a general matter) in thephotoresist layer(s) can be achieved by spinning the appropriate resistat predetermined rates (which can be found by reference to theliterature and manufacturer specifications, or empirically), in one ormore layers. (Well height typically may be selected in correspondencewith the lateral dimension of the sensor pixel, preferably for a nominal1:1-1.5:1 aspect ratio, height:width or diameter. Based onsignal-to-noise considerations, there is a relationship betweendimensions and the required data sampling rates to achieve a desiredlevel of performance. Thus there are a number of factors that will gointo selecting optimum parameters for a given application.)Alternatively, multiple layers of different photoresists may be appliedor another form of dielectric material may be deposited. Various typesof chemical vapor deposition may also be used to build up a layer ofmaterials suitable for microwell formation therein.

Once the photoresist layer (the singular form “layer” is used toencompass multiple layers in the aggregate, as well) is in place, theindividual wells (typically mapped to have either one or four ISFETsensors per well) may be generated by placing a mask (e.g., of chromium)over the resist-coated die and exposing the resist to cross-linking(typically UV) radiation. All resist exposed to the radiation (i.e.,where the mask does not block the radiation) becomes cross-linked and asa result will form a permanent plastic layer bonded to the surface ofthe chip (die). Unreacted resist (i.e., resist in areas which are notexposed, due to the mask blocking the light from reaching the resist andpreventing cross-linking) is removed by washing the chip in a suitablesolvent (i.e., developer) such as propyleneglycolmethylethylacetate(PGMEA) or other appropriate solvent. The resultant structure definesthe walls of the microwell array.

FIG. 30 shows an example of a layout for a portion of a chromium mask3010 for a one-sensor-per-well embodiment, corresponding to the portionof the die shown in FIG. 29. The grayed areas 3012, 3014 are those thatblock the UV radiation. The alignment marks in the white portions 3016on the bottom left quadrant of FIG. 30, within gray area 3012, are usedto align the layout of the wells with the ISFET sensors on the chipsurface. The array of circles 3014 in the upper right quadrant of themask block radiation from reaching the well areas, to leave unreactedresist which can be dissolved in forming the wells.

FIG. 31 shows a corresponding layout for the mask 3020 for a4-sensors-per-well embodiment. Note that the alignment pattern 3016 isstill used and that the individual well-masking circles 3014A in thearray 2910 now have twice the diameter as the wells 3014 in FIG. 30, foraccommodating four sensors per well instead of one sensor-per-well.

After exposure of the die/resist to the UV radiation, a second layer ofresist may be coated on the surface of the chip. This layer of resistmay be relatively thick, such as about 400-450 μm thick, typically. Asecond mask 3210 (FIG. 32), which also may be of chromium, is used tomask an area 3220 which surrounds the array, to build a collar or wall(or basin, using that term in the geological sense) 3310 of resist whichsurrounds the active array of sensors on substrate 3312, as shown inFIG. 33. In the particular example being described, the collar is 150 μmwider than the sensor array, on each side of the array, in the xdirection, and 9 μm wider on each side than the sensor array, in the ydirection. Alignment marks on mask 3210 (most not shown) are matched upwith the alignment marks on the first layer and the CMOS chip itself.

Other photolithographic approaches may be used for formation of themicrowell array, of course, the foregoing being only one example.

For example, contact lithography of various resolutions and with variousetchants and developers may be employed. Both organic and inorganicmaterials may be used for the layer(s) in which the microwells areformed. The layer(s) may be etched on a chip having a dielectric layerover the pixel structures in the sensor array, such as a passivationlayer, or the layer(s) may be formed separately and then applied overthe sensor array. The specific choice or processes will depend onfactors such as array size, well size, the fabrication facility that isavailable, acceptable costs, and the like.

Among the various organic materials which may be used in someembodiments to form the microwell layer(s) are the above-mentioned SU-8type of negative-acting photoresist, a conventional positive-actingphotoresist and a positive-acting photodefineable polyimide. Each hasits virtues and its drawbacks, well known to those familiar with thephotolithographic art.

Naturally, in a production environment, modifications will beappropriate.

Contact lithography has its limitations and it may not be the productionmethod of choice to produce the highest densities of wells—i.e., it mayimpose a higher than desired minimum pitch limit in the lateraldirections. Other techniques, such as a deep UV step-and-repeat process,are capable of providing higher resolution lithography and can be usedto produce small pitches and possibly smaller well diameters. Of course,for different desired specifications (e.g., numbers of sensors and wellsper chip), different techniques may prove optimal. And pragmaticfactors, such as the fabrication processes available to a manufacturer,may motivate the use of a specific fabrication method. While novelmethods are discussed, various aspects of the invention are limited touse of these novel methods.

Preferably the CMOS wafer with the ISFET array will be planarized afterthe final metallization process. A chemical mechanical dielectricplanarization prior to the silicon nitride passivation is suitable. Thiswill allow subsequent lithographic steps to be done on very flatsurfaces which are free of back-end CMOS topography.

By utilizing deep-UV step-and-repeat lithography systems, it is possibleto resolve small features with superior resolution, registration, andrepeatability. However, the high resolution and large numerical aperture(NA) of these systems precludes their having a large depth of focus. Assuch, it may be necessary, when using such a fabrication system, to usethinner photodefinable spin-on layers (i.e., resists on the order of 1-2μm rather than the thicker layers used in contact lithography) topattern transfer and then etch microwell features to underlying layer orlayers. High resolution lithography can then be used to pattern themicrowell features and conventional SiO₂ etch chemistries can beused—one each for the bondpad areas and then the microwell areas—havingselective etch stops; the etch stops then can be on aluminum bondpadsand silicon nitride passivation (or the like), respectively.Alternatively, other suitable substitute pattern transfer and etchprocesses can be employed to render microwells of inorganic materials.

Another approach is to form the microwell structure in an organicmaterial. For example, a dual-resist “soft-mask” process may beemployed, whereby a thin high-resolution deep-UV resist is used on topof a thicker organic material (e.g., cured polyimide or opposite-actingresist). The top resist layer is patterned. The pattern can betransferred using an oxygen plasma reactive ion etch process. Thisprocess sequence is sometimes referred to as the “portable conformablemask” (PCM) technique. See B. J. Lin et al., “Practicing the Novolacdeep-UV portable conformable masking technique”, Journal of VacuumScience and Technology 19, No. 4, 1313-1319 (1981); and A. Cooper et al,“Optimization of a photosensitive spin-on dielectric process for copperinductor coil and interconnect protection in RF SoC devices.”

Alternatively a “drill-focusing” technique may be employed, wherebyseveral sequential step-and-repeat exposures are done at different focaldepths to compensate for the limited depth of focus (DOF) ofhigh-resolution steppers when patterning thick resist layers. Thistechnique depends on the stepper NA and DOF as well as the contrastproperties of the resist material.

Another PCM technique may be adapted to these purposes, such as thatshown in U.S. patent application publication no. 2006/0073422 by Edwardset al. This is a three-layer PCM process and it is illustrated in FIG.33A. As shown there, basically six major steps are required to producethe microwell array and the result is quite similar to what contactlithography would yield.

In a first step, 3320, a layer of high contrast negative-actingphotoresist such as type Shipley InterVia Photodielectric Material 8021(IV8021) 3322 is spun on the surface of a wafer, which we shall assumeto be the wafer providing the substrate 3312 of FIG. 33 (in which thesensor array is fabricated), and a soft bake operation is performed.Next, in step 3324, a blocking anti-reflective coating (BARC) layer3326, is applied and soft baked. On top of this structure, a thin resistlayer 3328 is spun on and soft baked, step 3330, the thin layer ofresist being suitable for fine feature definition. The resist layer 3328is then patterned, exposed and developed, and the BARC in the exposedregions 3329, not protected any longer by the resist 3328, is removed,Step 3332. This opens up regions 3329 down to the uncured IV8021 layer.The BARC layer can now act like a conformal contact mask A blanketexposure with a flooding exposure tool, Step 3334, cross-links theexposed IV8021, which is now shown as distinct from the uncured IV8021at 3322. One or more developer steps 3338 are then performed, removingeverything but the cross-linked IV8021 in regions 3336. Regions 3336 nowconstitute the walls of the microwells.

Although as shown above, the wells bottom out (i.e. terminate) on thetop passivation layer of the ISFETs, it is believed that an improvementin ISFET sensor performance (i.e. such as signal-to-noise ratio) can beobtained if the active bead(s) is (are) kept slightly elevated from theISFET passivation layer. One way to do so is to place a spacer “bump”within the boundary of the pixel microwell. An example of how this couldbe rendered would be not etching away a portion of the layer-or-layersused to form the microwell structure (i.e. two lithographic steps toform the microwells—one to etch part way done, the other to pattern thebump and finish the etch to bottom out), by depositing andlithographically defining and etching a separate layer to form the“bump”, by using a permanent photo-definable material for the bump oncethe microwells are complete, or by forming the bump prior to forming themicrowell. The bump feature is shown as 3350 in FIG. 33B. An alternative(or additional) non-integrated approach is to load the wells with alayer or two of very small packing beads before loading the DNA-bearingbeads.

Using a 6 micron thick layer of tetra-methyl-ortho-silicate (TEOS) as aSiO₂-like layer for microwell formation, FIG. 33B-1 shows a scanningelectron microscope (SEM) image of a cross-section of a portion 3300A ofan array architecture as taught herein. Microwells 3302A are formed inthe TEOS layer 3304A. The wells extend about 4 μm into the 6 μm thicklayer. Typically, the etched well bottoms on an etch-stop material whichmay be, for example, an oxide, an organic material or other suitablematerial known in semiconductor processing for etch-stopping use. A thinlayer of etch stop material may be formed on top of a thicker layer ofpolyimide or other suitable dielectric, such that there is about 2 μm ofetch stop+polyimide between the well bottom and the Metal4 (M4) layer ofthe chip in which the extended gate electrode 3308A is formed for eachunderlying ISFET in the array. As labeled on the side, the CMOSmetallization layers M3, M2 and M1, which form lower level interconnectsand structures, are shown, with the ISFET channels being formed in theareas indicated by arrows 3310A.

In the orthogonal cross-sectional view (i.e., looking down from thetop), the wells may be formed in either round or square shape. Roundwells may improve bead capture and may obviate the need for packingbeads at the bottom or top of the wells.

The tapered slopes to the sides of the microwells also may be used toadvantage. Referring to FIG. 33B-2, if the beads 3320A have a diameterlarger than the bottom span across the wells, but small enough to fitinto the mouths of the wells, the beads will be spaced off the bottom ofthe wells due to the geometric constraints. For example, FIG. 33B-2illustrates the example of microwells that are square in cross-sectionas viewed from the top, 4 μm on a side, with 3.8 μm diameter beads 3320Aloaded. Experimentally and with some calculation, one may determinesuitable bead size and well dimension combinations. FIG. 33B-3 shows aportion of one 4 μm well loaded with a 2.8 μm diameter bead 3322A, whichobviously is relatively small and falls all the way to the bottom of thewell; a 4.0 μm diameter bead 3324A which is stopped from reaching thebottom by the sidewall taper of the well; and an intermediate-sized bead3326A of 3.6 μm diameter which is spaced from the well bottom by packingbeads 3328A. Clearly, bead size has to be carefully matched to themicrowell etch taper.

Thus, microwells can be fabricated by any high aspect ratiophoto-definable or etchable thin-film process, that can providerequisite thickness (e.g., about 4-10 um). Among the materials believedto be suitable are photosensitive polymers, deposited silicon dioxide,non-photosensitive polymer which can be etched using, for example,plasma etching processes, etc. In the silicon dioxide family, TEOS andsilane nitrous oxide (SILOX) appear suitable. The final structures aresimilar but the various materials present differing surface compositionsthat may cause the target biology or chemistry to react differently.

When the microwell layer is formed, it may be necessary to provide anetch stop layer so that the etching process does not proceed furtherthan desired. For example, there may be an underlying layer to bepreserved, such as a low-K dielectric. The etch stop material should beselected according to the application. SiC and SiN materials may besuitable, but that is not meant to indicate that other materials may notbe employed, instead. These etch-stop materials can also serve toenhance the surface chemistry which drives the ISFET sensor sensitivity,by choosing the etch-stop material to have an appropriate point of zerocharge (PZC). Various metal oxides may be suitable addition to silicondioxide and silicon nitride.

The PZCs for various metal oxides may be found in various texts, such as“Metal Oxides—Chemistry and Applications” by J. Fierro. We have learnedthat Ta₂O₅ may be preferred as an etch stop over Al₂O₃ because the PZCof Al₂O₃ is right at the pH being used (i.e., about 8.8) and, hence,right at the point of zero charge. In addition Ta₂O₅ has a highersensitivity to pH (i.e., mV/pH), another important factor in the sensorperformance. Optimizing these parameters may require judicious selectionof passivation surface materials.

Using thin metal oxide materials for this purpose (i.e., as an etch stoplayer) is difficult due to the fact of their being so thinly deposited(typically 200-500 A). A post-microwell fabrication metal oxidedeposition technique may allow placement of appropriate PZC metal oxidefilms at the bottom of the high aspect ratio microwells.

Electron-beam depositions of (a) reactively sputtered tantalum oxide,(b) non-reactive stoichiometric tantalum oxide, (c) tungsten oxide, or(d) Vanadium oxide may prove to have superior “down-in-well” coveragedue to the superior directionality of the deposition process.

The array typically comprises at least 100 microfluidic wells, each ofwhich is coupled to one or more chemFET sensors. Preferably, the wellsare formed in at least one of a glass (e.g., SiO₂), a polymericmaterial, a photodefinable material or a reactively ion etchable thinfilm material. Preferably, the wells have a width to height ratio lessthan about 1:1. Preferably the sensor is a field effect transistor, andmore preferably a chemFET. The chemFET may optionally be coupled to aPPi receptor. Preferably, each of the chemFETs occupies an area of thearray that is 10² microns or less.

In some embodiments, the invention encompasses a sequencing devicecomprising a semiconductor wafer device coupled to a dielectric layersuch as a glass (e.g., SiO₂), polymeric, photodefinable or reactive ionetchable material in which reaction chambers are formed. Typically, theglass, dielectric, polymeric, photodefinable or reactive ion etchablematerial is integrated with the semiconductor wafer layer. In someinstances, the glass, polymeric, photodefinable or reactive ion etchablelayer is non-crystalline. In some instances, the glass may be SiO₂. Thedevice can optionally further comprise a fluid delivery module of asuitable material such as a polymeric material, preferably an injectionmoldable material. More preferably, the polymeric layer ispolycarbonate.

In some embodiments, the invention encompasses a method formanufacturing a sequencing device comprising: using photolithography,generating wells in a glass, dielectric, photodefinable or reactivelyion etchable material on top of an array of transistors.

Yet another alternative when a CMOS or similar fabrication process isused for array fabrication is to form the microwells directly using theCMOS materials. That is, the CMOS top metallization layer forming thefloating gates of the ISFET array usually is coated with a passivationlayer that is about 1.3 μm thick. Microwells 1.3 μm deep can be formedby etching away the passivation material. For example, microwells havinga 1:1 aspect ratio may be formed, 1.3 μm deep and 1.3 μm across at theirtops. Modeling indicates that as the well size is reduced, in fact, theDNA concentration, and hence the SNR, increases. So, other factors beingequal, such small wells may prove desirable.

Mounting the Flow Cell (Fluidic Interface) to the Sensor Chip

The process of using the assembly of an array of sensors on a chipcombined with an array of microwells to sequence the DNA in a sample isreferred to as an “experiment.” Executing an experiment requires loadingthe wells with the DNA-bound beads and the flowing of several differentfluid solutions (i.e., reagents and washes) across the wells. A fluiddelivery system (e.g., valves, conduits, pressure source(s), etc.)coupled with a fluidic interface is needed which flows the varioussolutions across the wells in a controlled even flow with acceptablysmall dead volumes and small cross contamination between sequentialsolutions. Ideally, the fluidic interface to the chip (sometimesreferred to as a “flow cell”) would cause the fluid to reach allmicrowells at the same time. To maximize array speed, it is necessarythat the array outputs be available at as close to the same time aspossible. The ideal clearly is not possible, but it is desirable tominimize the differentials, or skews, of the arrival times of anintroduced fluid, at the various wells, in order to maximize the overallspeed of acquisition of all the signals from the array.

Flow cell designs of many configurations are possible; thus the systemand methods presented herein are not dependent on use of a specific flowcell configuration. It is desirable, though, that a suitable flow cellsubstantially conform to the following set of objectives:

-   -   have connections suitable for interconnecting with a fluidics        delivery system—e.g., via appropriately-sized tubing;    -   have appropriate head space above wells;    -   minimize dead volumes encountered by fluids;    -   minimize small spaces in contact with liquid but not quickly        swept clean by flow of a wash fluid through the flow cell (to        minimize cross contamination);    -   be configured to achieve uniform transit time of the flow over        the array;    -   generate or propagate minimal bubbles in the flow over the        wells;    -   be adaptable to placement of a removable reference electrode        inside or as close to the flow chamber as possible;    -   facilitate easy loading of beads;    -   be manufacturable at acceptable cost; and    -   be easily assembled and attached to the chip package.

Satisfaction of these criteria so far as possible will contribute tosystem performance positively. For example, minimization of bubbles isimportant so that signals from the array truly indicate the reaction ina well rather than being spurious noise.

Each of several example designs will be discussed, meeting thesecriteria in differing ways and degrees. In each instance, one typicallymay choose to implement the design in one of two ways: either byattaching the flow cell to a frame and gluing the frame (or otherwiseattaching it) to the chip or by integrating the frame into the flow cellstructure and attaching this unified assembly to the chip. Further,designs may be categorized by the way the reference electrode isintegrated into the arrangement. Depending on the design, the referenceelectrode may be integrated into the flow cell (e.g., form part of theceiling of the flow chamber) or be in the flow path (typically to theoutlet or downstream side of the flow path, after the sensor array).

A first example of a suitable experiment apparatus 3410 incorporatingsuch a fluidic interface is shown in FIGS. 34-37, the manufacture andconstruction of which will be discussed in greater detail below.

The apparatus comprises a semiconductor chip 3412 (indicated generally,though hidden) on or in which the arrays of wells and sensors areformed, and a fluidics assembly 3414 on top of the chip and deliveringthe sample to the chip for reading. The fluidics assembly includes aportion 3416 for introducing fluid containing the sample, a portion 3418for allowing the fluid to be piped out, and a flow chamber portion 3420for allowing the fluid to flow from inlet to outlet and along the wayinteract with the material in the wells. Those three portions areunified by an interface comprising a glass slide 3422 (e.g., ErieMicroarray Cat #C22-5128-M20 from Erie Scientific Company, Portsmouth,N.H., cut in thirds, each to be of size about 25 mm×25 mm).

Mounted on the top face of the glass slide are two fittings, 3424 and3426, such as nanoport fittings Part #N-333 from Upchurch Scientific ofOak Harbor, Wash. One port (e.g., 3424) serves as an inlet deliveringliquids from the pumping/valving system described below but not shownhere. The second port (e.g., 3426) is the outlet which pipes the liquidsto waste. Each port connects to a conduit 3428, 3432 such as flexibletubing of appropriate inner diameter. The nanoports are mounted suchthat the tubing can penetrate corresponding holes in the glass slide.The tube apertures should be flush with the bottom surface of the slide.

On the bottom of the glass slide, flow chamber 3420 may comprise variousstructures for promoting a substantially laminar flow across themicrowell array. For example, a series of microfluidic channels fanningout from the inlet pipe to the edge of the flow chamber may be patternedby contact lithography using positive photoresists such as SU-8photoresist from MicroChem Corp. of Newton, Mass. Other structures willbe discussed below.

The chip 3412 will in turn be mounted to a carrier 3430, for packagingand connection to connector pins 3432.

For ease of description, to discuss fabrication starting with FIG. 38 weshall now consider the glass slide 3422 to be turned upside downrelative to the orientation it has in FIGS. 34-37.

A layer of photoresist 3810 is applied to the “top” of the slide (whichwill become the “bottom” side when the slide and its additional layersis turned over and mounted to the sensor assembly of ISFET array withmicrowell array on it). Layer 3810 may be about 150 μm thick in thisexample, and it will form the primary fluid carrying layer from the endof the tubing in the nanoports to the edge of the sensor array chip.Layer 3810 is patterned using a mask such as the mask 3910 of FIG. 39(“patterned’ meaning that a radiation source is used to expose theresist through the mask and then the non-plasticized resist is removed).The mask 3910 has radiation-transparent regions which are shown as whiteand radiation-blocking regions 3920, which are shown in shading. Theradiation-blocking regions are at 3922-3928. The region 3926 will form achannel around the sensor assembly; it is formed about 0.5 mm inside theouter boundary of the mask 3920, to avoid the edge bead that is typical.The regions 3922 and 3924 will block radiation so that correspondingportions of the resist are removed to form voids shaped as shown. Eachof regions 3922, 3924 has a rounded end dimensioned to receive an end ofa corresponding, one of the tubes 3428, 3432 passing through acorresponding nanoport 3424, 3426. From the rounded end, the regions3922, 3924 fan out in the direction of the sensor array to allow theliquid to spread so that the flow across the array will be substantiallylaminar. The region 3928 is simply an alignment pattern and may be anysuitable alignment pattern or be replaced by a suitable substitutealignment mechanism. Dashed lines on FIG. 38 have been provided toillustrate the formation of the voids 3822 and 3824 under mask regions3922 and 3924.

A second layer of photoresist is formed quite separately, not on theresist 3810 or slide 3422. Preferably it is formed on a flat, flexiblesurface (not shown), to create a peel-off, patterned plastic layer. Asshown in FIG. 40, this second layer of photoresist may be formed using amask such as mask 4010, which will leave on the flexible substrate,after patterning, the border under region 4012, two slits under regions4014, 4016, whose use will be discussed below, and alignment marksproduced by patterned regions 4018 and 4022. The second layer ofphotoresist is then applied to the first layer of photoresist using onealignment mark or set of alignment marks, let's say produced by pattern4018, for alignment of these layers. Then the second layer is peeledfrom its flexible substrate and the latter is removed.

The other alignment mark or set of marks produced by pattern 4022 isused for alignment with a subsequent layer to be discussed.

The second layer is preferably about 150 μm deep and it will cover thefluid-carrying channel with the exception of a slit about 150 μm long ateach respective edge of the sensor array chip, under slit-formingregions 4014 and 4016.

Once the second layer of photoresist is disposed on the first layer, athird patterned layer of photoresist is formed over the second layer,using a mask such as mask 4110, shown in FIG. 41. The third layerprovides a baffle member under region 4112 which is as wide as thecollar 3310 on the sensor chip array (see FIG. 33) but about 300 μmnarrower to allow overlap with the fluid-carrying channel of the firstlayer. The third layer may be about 150 μm thick and will penetrate thechip collar 3310, toward the floor of the basin formed thereby, by 150μm. This configuration will leave a headspace of about 300 μm above thewells on the sensor array chip. The liquids are flowed across the wellsalong the entire width of the sensor array through the 150 μm slitsunder 4014, 4016.

FIG. 36 shows a partial sectional view, in perspective, of theabove-described example embodiment of a microfluidics and sensorassembly, also depicted in FIGS. 34 and 35, enlarged to make morevisible the fluid flow path. (A further enlarged schematic of half ofthe flow path is shown in FIG. 37.) Here, it will be seen that fluidenters via the inlet pipe 3428 in inlet port 3424. At the bottom of pipe3428, the fluid flows through the flow expansion chamber 3610 formed bymask area 3922, that the fluid flows over the collar 3310 and then downinto the bottom 3320 of the basin, and across the die 3412 with itsmicrowell array. After passing over the array, the fluid then takes avertical turn at the far wall of the collar 3310 and flows over the topof the collar to and across the flow concentration chamber 3612 formedby mask area 3924, exiting via outlet pipe 3432 in outlet port 3426.Part of this flow, from the middle of the array to the outlet, may beseen also in the enlarged diagrammatic illustration of FIG. 37, whereinthe arrows indicate the flow of the fluid.

The fluidics assembly may be secured to the sensor array chip assemblyby applying an adhesive to parts of mating surfaces of those twoassemblies, and pressing them together, in alignment.

Though not illustrated in FIGS. 34-36, the reference electrode may beunderstood to be a metallization 3710, as shown in FIG. 37, at theceiling of the flow chamber.

Another way to introduce the reference electrode is shown in FIG. 42.There, a hole 4210 is provided in the ceiling of the flow chamber and agrommet 4212 (e.g., of silicone) is fitted into that hole, providing acentral passage or bore through which a reference electrode 4220 may beinserted. Baffles or other microfeatures (not shown in FIG. 42 butdiscussed below in connection with FIG. 42A) may be patterned into theflow channel to promote laminar flow over the microwell array.

Achieving a uniform flow front and eliminating problematic flow pathareas is desirable for a number of reasons. One reason is that very fasttransition of fluid interfaces within the system's flow cell is desiredfor many applications, particularly gene sequencing. In other words, anincoming fluid must completely displace the previous fluid in a shortperiod of time. Uneven fluid velocities and diffusion within the flowcell, as well as problematic flow paths, can compete with thisrequirement. Simple flow through a conduit of rectangular cross sectioncan exhibit considerable disparity of fluid velocity from regions nearthe center of the flow volume to those adjacent the sidewalls, onesidewall being the top surface of the microwell layer and the fluid inthe wells. Such disparity leads to spatially and temporally largeconcentration gradients between the two traveling fluids. Further,bubbles are likely to be trapped or created in stagnant areas like sharpcorners interior the flow cell. (The surface energy (hydrophilic vs.hydrophobic) can significantly affect bubble retention. Avoidance ofsurface contamination during processing and use of a surface treatmentto create a more hydrophilic surface should be considered if theas-molded surface is too hydrophobic.) Of course, the physicalarrangement of the flow chamber is probably the factor which mostinfluences the degree of uniformity achievable for the flow front.

One approach is to configure the flow cross section of the flow chamberto achieve flow rates that vary across the array width so that thetransit times are uniform across the array. For example, the crosssection of the diffuser (i.e., flow expansion chamber) section 3416,3610 may be made as shown at 4204A in FIG. 42A, instead of simply beingrectangular, as at 4204A. That is, it may have a curved (e.g., concave)wall. The non-flat wall 4206A of the diffuser can be the top or thebottom. Another approach is to configure the effective path lengths intothe array so that the total path lengths from entrance to exit over thearray are essentially the same. This may be achieved, for example, byplacing flow-disrupting features such as cylinders or other structuresoriented normal to the flow direction, in the path of the flow. If theflow chamber has as a floor the top of the microwell array and as aceiling an opposing wall, these flow-disrupting structures may beprovided either on the top of the microwell layer or on (or in) theceiling wall. The structures must project sufficiently into the flow tohave the desired effect, but even small flow disturbances can havesignificant impact. Turning to FIGS. 42B-42F, there are showndiagrammatically some examples of such structures. In FIG. 42B, on thesurface of microwell layer 4210B there are formed a series ofcylindrical flow disruptors 4214B extending vertically toward the flowchamber ceiling wall 4212B, and serving to disturb laminar flow for thefluid moving in the direction of arrow A. FIG. 42C depicts a similararrangement except that the flow disruptors 4216C have rounded tops andappear more like bumps, perhaps hemispheres or cylinders with sphericaltops. By contrast, in FIG. 42D, the flow disruptors 4218D protrude, ordepend, from the ceiling wall 4212B of the flow chamber. Only one columnof flow disruptors is shown but it will be appreciated that a pluralityof more or less parallel columns typically would be required. Forexample, FIG. 42E shows several columns 4202E of such flow disruptors(projecting outwardly from ceiling wall 4212B (though the orientation isupside down relative to FIGS. 42B-42D). The spacing between thedisruptors and their heights may be selected to influence the distanceover which the flow profile becomes parabolic, so that transit timeequilibrates.

Another configuration, shown in FIGS. 42F and 42F1, involves the use ofsolid, beam-like projections or baffles 4220F as disruptors. Thisconcept may be used to form a ceiling member for the flow chamber. Suchan arrangement encourages more even fluid flow and significantly reducesfluid displacement times as compared with a simple rectangularcross-section without disruptor structure. Further, instead of fluidentry to the array occurring along one edge, fluid may be introduced atone corner 4242F, through a small port, and may exit from the oppositecorner, 4244F, via a port in fluid communication with that corner area.The series of parallel baffles 4220F separates the flow volume betweeninput and outlet corners into a series of channels. The lowest fluidresistant path is along the edge of the chip, perpendicular to thebaffles. As incoming liquid fills this channel, the fluid is thendirected between the baffles to the opposite side of the chip. Thechannel depth between each baffle pair preferably is graded across thechip, such that the flow is encouraged to travel toward the exit portthrough the farthest channel, thereby evening the flow front between thebaffles. The baffles extend downwardly nearly to the chip (i.e.,microwell layer) surface, but because they are quite thin, fluid candiffuse under them quickly and expose the associated area of the arrayassembly.

FIGS. 42F2-42F8 illustrate an example of a single-piece,injection-molded (preferably of polycarbonate) flow cell member 42F200which may be used to provide baffles 4220F, a ceiling to the flowchamber, fluid inlet and outlet ports and even the reference electrode.FIG. 42F7 shows an enlarged view of the baffles on the bottom of member42F200 and the baffles are shown as part of the underside of member42F200 in FIG. 42F6. As it is difficult to form rectangular features insmall dimensions by injection molding, the particular instance of thesebaffles, shown as 4220F′, are triangular in cross section.

In FIG. 42F2, there is a top, isometric view of member 42F200 mountedonto a sensor array package 42F300, with a seal 42F202 formed betweenthem and contact pins 42F204 depending from the sensor array chippackage. FIGS. 42F3 and 42F4 show sections, respectively, throughsection lines H-H and I-I of FIG. 42F5, permitting one to see inrelationship the sensor array chip 42F250, the baffles 4220F′ and fluidflow paths via inlet 42F260 and outlet 42F270 ports.

By applying a metallization to bottom 42F280 of member 42F200, thereference electrode may be formed.

Various other locations and approaches may be used for introducing fluidflow into the flow chamber, as well. In addition to embodiments in whichfluid may be introduced across the width of an edge of the chip assembly42F1, as in FIGS. 57-58, for example, or fluid may be introduced at onecorner of the chip assembly, as in FIG. 42F1. Fluid also may beintroduced, for example, as in FIGS. 42G and 42H, where fluid is flowedthrough an inlet conduit 4252G to be discharged adjacent and toward thecenter of the chip, as at 4254G, and flowed radially outwardly from theintroduction point.

FIGS. 42I and 42J in conjunction with FIGS. 42G and 42H depict incross-section an example of such a structure and its operation. Incontrast with earlier examples, this embodiment contains an additionalelement, a diaphragm valve, 4260I. Initially, as shown in FIG. 42H, thevalve 4260I is open, providing a path via conduit 4262I to a wastereservoir (not shown). The open valve provides a low impedance flow tothe waste reservoir or outlet. Air pressure is then applied to thediaphragm valve, as in FIG. 42J, closing the low impedance path andcausing the fluid flow to continue downwardly through central bore 4264Jin member 4266J which forms the ceiling of the flow chamber, and acrossthe chip (sensor) assembly. The flow is collected by the channels at theedges of the sensor, as described above, and exits to the waste outputvia conduit 4268J.

A variation on this idea is depicted in FIGS. 42K-42M, which show fluidbeing introduced not at the center of the chip assembly, but at onecorner, 4272K, instead. It flows across the chip 3412 as symbolicallyindicated by lines 4274K and is removed at the diagonally opposingcorner, 4276K. The advantage of this concept is that it all buteliminates any stagnation points. It also has the advantage that thesensor array can be positioned vertically so that the flow is introducedat the bottom and removed at the top to aid in the clearance of bubbles.This type of embodiment, by the way, may be considered as one quadrantof the embodiments with the flow introduced in the center of the array.An example of an implementation with a valve 4278L closed and shuntingflow to the waste outlet or reservoir is shown in FIG. 42L. The maindifference with respect to the embodiment of FIGS. 42I and 42J is thatthe fluid flow is introduced at a corner of the array rather than at itscenter.

In all cases, attention should be given to assuring a thorough washingof the entire flow chamber, along with the microwells, between reagentcycles. Flow disturbances may exacerbate the challenge of fully cleaningout the flow chamber.

Flow disturbances may also induce or multiply bubbles in the fluid. Abubble may prevent the fluid from reaching a microwell, or delay itsintroduction to the microwell, introducing error into the microwellreading or making the output from that microwell useless in theprocessing of outputs from the array. Thus, care should be taken inselecting configurations and dimensions for the flow disruptor elementsto manage these potential adverse factors. For example, a tradeoff maybe made between the heights of the disruptor elements and the velocityprofile change that is desired.

FIGS. 43-44 show another alternative flow cell design, 4310. This designrelies on the molding of a single plastic piece or member 4320 to beattached to the chip to complete the flow cell. The connection to thefluidic system is made via threaded connections tapped into appropriateholes in the plastic piece at 4330 and 4340. Or, if the member 4320 ismade of a material such as polydimethylsiloxane (PDMS), the connectionsmay be made by simply inserting the tubing into an appropriately sizedhole in the member 4320. A vertical cross section of this design isshown in FIGS. 43-44. This design may use an overhanging plastic collar4350 (which may be a solid wall as shown or a series of depending,spaced apart legs forming a downwardly extending fence-like wall) toenclose the chip package and align the plastic piece with the chippackage, or other suitable structure, and thereby to alignment the chipframe with the flow cell forming member 4320. Liquid is directed intothe flow cell via one of apertures 4330, 4340, thence downwardly towardsthe flow chamber.

In the illustrated embodiment, the reference electrode is introduced tothe top of the flow chamber via a bore 4325 in the member 4320. Theplacement of the removable reference electrode is facilitated by asilicone sleeve 4360 and an epoxy stop ring 4370 (see the blow-up ofFIG. 44). The silicone sleeve provides a tight seal and the epoxy stopring prevent the electrode from being inserted too far into the flowcell. Of course, other mechanisms may be employed for the same purposes,and it may not be necessary to employ structure to stop the electrode.And if a material such as PDMS is used for member 4320, the materialitself may form a watertight seal when the electrode is inserted,obviating need for the silicone sleeve.

FIGS. 45 and 46 show a similar arrangement except that member 4510 lacksa bore for receiving a reference electrode. Instead, the referenceelectrode 4515 is formed on or affixed to the bottom of central portion4520 and forms at least part of the flow chamber ceiling. For example, ametallization layer may be applied onto the bottom of central portion4520 before member 4510 is mounted onto the chip package.

FIGS. 47-48 show another example, which is a variant of the embodimentshown in FIGS. 43-44, but wherein the frame is manufactured as part ofthe flow cell rather attaching a flow port structure to a framepreviously attached to the chip surface. In designs of this type,assembly is somewhat more delicate since the wirebonds to the chip arenot protected by the epoxy encapsulating the chip. The success of thisdesign is dependent on the accurate placement and secure gluing of theintegrated “frame” to the surface of the chip. A counterpart embodimentto that of FIGS. 45-46, with the reference electrode 4910 on the ceilingof the flow chamber, and with the frame manufactured as part of the flowcell, is shown in FIGS. 49-50.

Yet another alternative for a fluidics assembly, as shown in FIGS.51-52, has a fluidics member 5110 raised by about 5.5 mm on stand-offs5120 from the top of the chip package 5130. This allows for an operatorto visually inspect the quality of the bonding between plastic piece5140 and chip surface and reinforce the bonding externally if necessary.

Some of the foregoing alternative embodiments also may be implemented ina hybrid plastic/PDMS configuration. For example, as shown in FIGS.53-54, a plastic part 5310 may make up the frame and flow chamber,resting on a PDMS “base” portion 5320. The plastic part 5310 may alsoprovides a region 5330 to the array, for expansion of the fluid flowfrom the inlet port; and the PDMS part may then include communicatingslits 5410, 5412 through which liquids are passed from the PDMS part toand from the flow chamber below.

The fluidic structure may also be made from glass as discussed above,such as photo-definable (PD) glass. Such a glass may have an enhancedetch rate in hydrofluoric acid once selectively exposed to UV light andfeatures may thereby be micromachined on the top-side and back-side,which when glued together can form a three-dimensional low aspect ratiofluidic cell.

An example is shown in FIG. 55. A first glass layer or sheet 5510 hasbeen patterned and etched to create nanoport fluidic holes 5522 and 5524on the top-side and fluid expansion channels 5526 and 5528 on theback-side. A second glass layer or sheet 5530 has been patterned andetched to provide downward fluid input/output channels 5532 and 5534, ofabout 300 μm height (the thickness of the layer). The bottom surface oflayer 5530 is thinned to the outside of channels 5532 and 5534, to allowthe layer 5530 to rest on the chip frame and protrusion area 5542 to beat an appropriate height to form the top of the flow channel. Two glasslayers, or wafers, and four lithography steps required. Both wafersshould be aligned and bonded (e.g., with an appropriate glue, not shown)such that the downward fluid input/output ports are aligned properlywith the fluid expansion channels. Alignment targets may be etched intothe glass to facilitate the alignment process.

Nanoports may be secured over the nanoport fluidic holes to facilitateconnection of input and output tubing.

A central bore 5550 may be etched through the glass layers for receivinga reference electrode, 5560. The electrode may be secured and sealed inplace with a silicone collar 5570 or like structure; or the electrodemay be equipped integrally with a suitable washer for effecting the samepurpose.

By using glass materials for the two-layer fluidic cell, the referenceelectrode may also be a conductive layer or pattern deposited on thebottom surface of the second glass layer (not shown). Or, as shown inFIG. 56, the protrusion region may be etched to form a permeable glassmembrane 5610 on the top of which is coated a silver (or other material)thin-film 5620 to form an integrated reference electrode. A hole 5630may be etched into the upper layer for accessing the electrode and ifthat hole is large enough, it can also serve as a reservoir for a silverchloride solution. Electrical connection to the thin-film silverelectrode may be made in any suitable way, such as by using a clip-onpushpin connector or alternatively wirebonded to the ceramic ISFETpackage.

Another alternative is to integrate the reference electrode to thesequencing chip/flow cell by using a metalized surface on the ceiling ofthe flow chamber—i.e., on the underside of the member forming theceiling of the fluidic cell. An electrical connection to the metalizedsurface may be made in any of a variety of ways, including, but notlimited to, by means of applying a conductive epoxy to the ceramicpackage seal ring that, in turn, may be electrically connected through avia in the ceramic substrate to a spare pin at the bottom of the chippackage. Doing this would allow system-level control of the referencepotential in the fluid cell by means of inputs through the chip socketmount to the chip's control electronics.

By contrast, an externally inserted electrode requires extra fluidtubing to the inlet port, which requires additional fluid flow betweencycles.

Ceramic pin grid array (PGA) packaging may be used for the ISFET array,allowing customized electrical connections between various surfaces onthe front face with pins on the back.

The flow cell can be thought of as a “lid” to the ISFET chip and itsPGA. The flow cell, as stated elsewhere, may be fabricated of manydifferent materials. Injection molded polycarbonate appears to be quitesuitable. A conductive metal (e.g., gold) may be deposited using anadhesion layer (e.g., chrome) to the underside of the glow cell roof(the ceiling of the flow chamber). Appropriate low-temperature thin-filmdeposition techniques preferably are employed in the deposition of themetal reference electrode due to the materials (e.g., polycarbonate) andlarge step coverage topography at the bottom-side of the fluidic cell(i.e., the frame surround of ISFET array). One possible approach wouldbe to use electron-beam evaporation in a planetary system.

The active electrode area is confined to the central flow chamber insidethe frame surround of the ISFET array, as that is the only metalizedsurface that would be in contact with the ionic fluid during sequencing.

Once assembly is complete—conductive epoxy (e.g., Epo-Tek H₂₀E orsimilar) may be dispensed on the seal ring with the flow cell aligned,placed, pressed and cured—the ISFET flow cell is ready for operationwith the reference potential being applied to the assigned pin of thepackage.

The resulting fluidic system connections to the ISFET device thusincorporate shortened input and output fluidic lines, which isdesirable.

Still another example embodiment for a fluidic assembly is shown inFIGS. 57-58. This design is limited to a plastic piece 5710 whichincorporates the frame and is attached directly to the chip surface, andto a second piece 5720 which is used to connect tubing from the fluidicsystem and similarly to the PDMS piece discussed above, distributes theliquids from the small bore tube to a wide flat slit. The two pieces areglued together and multiple (e.g., three) alignment markers (not shown)may be used to precisely align the two pieces during the gluing process.A hole may be provided in the bottom plate and the hole used to fill thecavity with an epoxy (for example) to protect the wirebonds to the chipand to fill in any potential gaps in the frame/chip contact. In theillustrated example, the reference electrode is external to the flowcell (downstream in the exhaust stream, through the outlet port—seebelow), though other configurations of reference electrode may, ofcourse, be used.

Still further examples of flow cell structures are shown in FIGS. 59-60.FIG. 59A comprises eight views (A-H) of an injection molded bottomlayer, or plate, 5910, for a flow cell fluidics interface, while FIG.59B comprises seven views (A-G) of a mating, injection molded top plate,or layer, 5950. The bottom of plate 5910 has a downwardly depending rim5912 configured and arranged to enclose the sensor chip and an upwardlyextending rim 5914 for mating with the top plate 5610 along its outeredge. To form two fluid chambers (an inlet chamber and an outletchamber) between them. A stepped, downwardly depending portion 5960 oftop plate 5950, separates the input chamber from the output chamber. Aninlet tube 5970 and an outlet tube 5980 are integrally molded with therest of top plate 5950. From inlet tube 5970, which empties at the smallend of the inlet chamber formed by a depression 5920 in the top of plate5910, to the outlet edge of inlet chamber fans out to direct fluidacross the whole array.

Whether glass or plastic or other material is used to form the flowcell, it may be desirable, especially with larger arrays, to include inthe inlet chamber of the flow cell, between the inlet conduit and thefront edge of the array, not just a gradually expanding (fanning out)space, but also some structure to facilitate the flow across the arraybeing suitably laminar. Using the bottom layer 5990 of an injectionmolded flow cell as an example, one example type of structure for thispurpose, shown in FIG. 59C, is a tree structure 5992 of channels fromthe inlet location of the flow cell to the front edge of the microwellarray or sensor array, which should be understood to be under the outletside of the structure, at 5994.

The above-described systems typically utilize a laminar fluid flowsystem. In part, the fluid flow system preferably includes a flowchamber formed by the sensor chip and a single piece, injection moldedmember comprising inlet and outlet ports and mountable over the chip toestablish the flow chamber. The surface of such member interior to thechamber is preferably formed to facilitate a desired expedient fluidflow, as described herein.

In some embodiments, the invention encompasses an apparatus fordetection of pH comprising a laminar fluid flow system. Preferably, theapparatus is used for sequencing a plurality of nucleic acid templatespresent in an array.

The apparatus typically includes a fluidics assembly comprising a membercomprising one or more apertures for non-mechanically directing a fluidto flow to an array of at least 100K (100 thousand), 500K (500thousand), or 1 M (1 million) microfluidic reaction chambers such thatthe fluid reaches all of the microfluidic reaction chambers at the sametime or substantially the same time. Typically, the fluid flow isparallel to the sensor surface. Typically, the assembly has a Reynoldsnumber of less than 1000, 500, 200, 100, 50, 20, or 10. Preferably, themember further comprises a first aperture for directing fluid towardsthe sensor array and a second aperture for directing fluid away from thesensor array.

In some embodiments, the invention encompasses a method for directing afluid to a sensor array comprising: providing a fluidics assemblycomprising an aperture fluidly coupling a fluid source to the sensorarray; and non-mechanically directing a fluid to the sensor array. By“non-mechanically” it is meant that the fluid is moved under pressurefrom a gaseous pressure source, as opposed to a mechanical pump.

In some embodiments, the invention encompasses an array of wells, eachof which is coupled to a lid having an inlet port and an outlet port anda fluid delivery system for delivering and removing fluid from saidinlet and outlet ports non-mechanically.

In some embodiments, the invention encompasses a method for sequencing abiological polymer such as a nucleic acid utilizing the above-describedapparatus, comprising: directing a fluid comprising a monomer to anarray of reaction chambers wherein the fluid has a fluid flow Reynoldsnumber of at most 2000, 1000, 200, 100, 50, or 20. The method mayoptionally further comprise detecting a pH or a change in pH from eachsaid reaction chamber. This is typically detected by ion diffusion tothe sensor surface. There are various other ways of providing a fluidicsassembly for delivering an appropriate fluid flow across the microwelland sensor array assembly, and the forgoing examples are thus notintended to be exhaustive.

Reference Electrode

Commercial flow-type fluidic electrodes, such as silver chlorideproton-permeable electrodes, may be inserted in series in a fluidic lineand are generally designed to provide a stable electrical potentialalong the fluidic line for various electrochemical purposes. In theabove-discussed system, however, such a potential must be maintained atthe fluidic volume in contact with the microwell ISFET chip. Withconventional silver chloride electrodes, it has been found difficult,due to an electrically long fluidic path between the chip surface andthe electrode (through small channels in the flow cell), to achieve astable potential. This led to reception of noise in the chip'selectronics. Additionally, the large volume within the flow cavity ofthe electrode tended to trap and accumulate gas bubbles that degradedthe electrical connection to the fluid. With reference to FIG. 60, asolution to this problem has been found in the use of a stainless steelcapillary tube electrode 6010, directly connected to the chip's flowcell outlet port 6020 and connected to a voltage source (not shown)through a shielded cable 6030. The metal capillary tube 6010 has a smallinner diameter (e.g., on the order of 0.01″) that does not trap gas toany appreciable degree and effectively transports fluid and gas likeother microfluidic tubing. Also, because the capillary tube can bedirectly inserted into the flow cell port 6020, it close to the chipsurface, reducing possible electrical losses through the fluid. Thelarge inner surface area of the capillary tube (typically about 2″ long)may also contribute to its high performance. The stainless steelconstruction is highly chemically resistant, and should not be subjectto electrochemical effects due to the very low electrical current use inthe system (<1 μA). A fluidic fitting 6040 is attached to the end of thecapillary that is not in the flow cell port, for connection to tubing tothe fluid delivery and removal subsystem.

Fluidics System

A complete system for using the sensor array will include suitable fluidsources, valving and a controller for operating the valving to lowreagents and washes over the microarray or sensor array, depending onthe application. These elements are readily assembled from off-the-shelfcomponents, with and the controller may readily be programmed to performa desired experiment.

It should be understood that the readout at the chemFET may be currentor voltage (and change thereof) and that any particular reference toeither readout is intended for simplicity and not to the exclusion ofthe other readout. Therefore any reference in the following text toeither current or voltage detection at the chemFET should be understoodto contemplate and apply equally to the other readout as well. Inimportant embodiments, the readout reflects a rapid, transient change inconcentration of an analyte. The concentration of more than one analytemay be detected at different times. In some instances, such measurementsare to be contrasted with methods that focus on steady stateconcentration measurements.

Biological and Chemical Reactions

As already discussed, the apparatus, systems and methods of theinvention can be used to detect and/or monitor interactions betweenvarious entities. These interactions include biological and chemicalreactions and may involve enzymatic reactions and/or non-enzymaticinteractions such as but not limited to binding events. As an example,the invention contemplates monitoring enzymatic reactions in whichsubstrates and/or reagents are consumed and/or reaction intermediates,byproducts and/or products are generated. An example of a reaction thatcan be monitored according to the invention is a nucleic acid synthesismethod such as one that provides information regarding nucleic acidsequence. This reaction will be discussed in greater detail herein.

Nucleic Acid Sequencing

In the context of a sequencing reaction, the apparatus and systemprovided herein is able to detect nucleotide incorporation based onchanges in the chemFET current and/or voltage, as those latterparameters are interrelated. Current changes may be the result of one ormore of the following events either singly or some combination thereof:generation of hydrogen (and concomitant changes in pH for example in thepresence of low strength buffer or no buffer), generation of PPi,generation of Pi (e.g., in the presence of pyrophosphatase), increasedcharge of nucleic acids attached to the chemFET surface, and the like.

As discussed herein, the invention contemplates methods for determiningthe nucleotide sequence of a nucleic acid. Such methods involve thesynthesis of a new nucleic acid (e.g., using a primer that is hybridizedto a template nucleic acid or a self-priming template, as will beappreciated by those of ordinary skill), based on the sequence of atemplate nucleic acid. That is, the sequence of the newly synthesizednucleic acid is complimentary to the sequence of the template nucleicacid and therefore knowledge of sequence of the newly synthesizednucleic acid yields information about the sequence of the templatenucleic acid.

More specifically, knowledge of the sequence of the newly synthesizednucleic acid is obtained by determining whether a known nucleotide hasbeen incorporated into the newly synthesized nucleic acid and, if so,how many of such known nucleotides have been incorporated. Importantly,the order in which the known nucleotides are added to the reactionmixture is known and thus the order of incorporated nucleotides (if any)is also known. In an illustrative embodiment, a template hybridized to aprimer is contacted with a first pool of identical known nucleotides(e.g., dATP) in the presence of polymerase. If the next availableposition on the template is a thymidine residue, then the dATP isincorporated into the primed nucleic acid strand and a signal isdetected for example based on hydrogen release. If the next availableposition is not a thymidine residue, then the dATP will not incorporateand no signal will be detected because no hydrogen will be released. Ifthe next available position and one or more contiguous positionsthereafter are thymidine residues, then a corresponding number of dATPwill be incorporated and a signal commensurate with the number ofnucleotides incorporated will be detected. The reaction well or chamberis then washed to remove unincorporated nucleotides and releasedhydrogen, following which another pool of identical known nucleotides(e.g., dCTP) is added. The process is repeated until all fournucleotides are separately added to the reaction well (i.e., one cycle),and then the cycles are repeated. The cycles may be repeated for 50times, 100 times, 200 times, 300 times, 400 times, 500 times, 750 times,or more, depending on the length of sequence information desired.

Nucleotide incorporation can be monitored in a number of ways, includingthe production of products such as PPi, Pi and/or H⁺. The incorporationof a dNTP into the nucleic acid strand releases PPi which can then behydrolyzed to two orthophosphates (Pi) and one hydrogen ion (FIG. 61A).The generation of the hydrogen ion therefore can be detected as anindicator of nucleotide incorporation. Alternatively, Pi may be detecteddirectly or indirectly.

Alternatively, when templates or primers are attached to the sensorsurface, nucleotide incorporation is detected based on an increase incharge (typically, negative charge) of the template, primer ortemplate/primer complex. Templates may be bound to the chemFET surfaceor they may be hybridized to primers that are bound to the chemFETsurface. Primers hybridized to the templates can be extended in thepresence of polymerase and one or a combination of known nucleotides.Nucleotide incorporation is detected by increases in charge at thechemFET surface that result from the addition of phosphodiester backbonelinkages that carry negative charges. Thus, with each successiveaddition of a nucleotide, the negative charge of the immobilized nucleicacid increases, and this increase can be detected by the chemFET. Thenumber of nucleotide incorporations that can be detected in this mannermay be at least 10, at least 20, at least 30, at least 40, at least 50,at least 60, at least 70, at least 80, at least 90, at least 100, ormore. The invention contemplates that in this instance nucleotideincorporation can be detected by measuring change in charge at thechemFET surface as well as released hydrogen ions that come into contactwith the chemFET.

Any and all of these events (and more as described herein) may bedetected at the chemFET thereby causing a current change that correlateswith nucleotide incorporation.

The systems described herein can be used for sequencing nucleic acidswithout optical detection. Preferably, at least 10⁶ base pairs aresequenced per hour, more preferably at least 10⁷ base pairs aresequenced per hour, and most preferably at least 10⁸ base pairs aresequenced per hour using the above-described method. Thus, the methodmay be used to sequence an entire human genome within about 24 hours,more preferably within about 20 hours, even more preferably within about15 hours, even more preferably within about 10 hours, even morepreferably within about 5 hours, and most preferably within about 1hour. These rates may be achieved using multiple ISFET arrays as shownherein, and processing their outputs in parallel.

pH Based Nucleic Acid Sequencing

Reduced Buffering

Certain aspects of the invention therefore relate to detecting hydrogenions released as a function of nucleotide incorporation and in someembodiments as a function of nucleotide excision. It is important inthese and various other aspects to detect as many released hydrogen ionsas possible in order to achieve as high a signal (and/or a signal tonoise ratio) as possible. Strategies for increasing the number ofreleased protons that are ultimately detected by the chemFET surfaceinclude without limitation limiting interaction of released protons withreactive groups in the well, choosing a material from which tomanufacture the well in the first instance that is relatively inert toprotons, preventing released protons from exiting the well prior todetection at the chemFET, and increasing the copy number of templatesper well (in order to amplify the signal from each nucleotideincorporation), among others.

Some instances of the invention employ an environment, including areaction solution, that is minimally buffered, if at all. Buffering canbe contributed by the components of the solution or by the solidsupports in contact with such solution. A solution having no or lowbuffering capacity (or activity) is one in which changes in hydrogen ionconcentration on the order of at least about +/−0.005 pH units, at leastabout +/−0.01, at least about +/−0.015, at least about +/−0.02, at leastabout +/−0.03, at least about +/−0.04, at least about +/−0.05, at leastabout +/−0.10, at least about +/−0.15, at least about +/−0.20, at leastabout +/−0.25, at least about +/−0.30, at least about +/−0.35, at leastabout +/−0.45, at least about +/−0.50, or more are detectable (e.g.,using the chemFET sensors described herein). In some embodiments, the pHchange per nucleotide incorporation is on the order of about 0.005. Insome embodiments, the pH change per nucleotide incorporation is adecrease in pH. Reaction solutions that have no or low bufferingcapacity may contain no or very low concentrations of buffer, or may useweak buffers.

A buffer is an ionic molecule (or a solution comprising an ionicmolecule) that resists, to varying extents, changes in pH. Buffersinclude without limitation Tris, tricine, phosphate, boric acid, borate,acetate, morpholine, citric acid, carbonic acid, and phosphoric acid.The strength of a buffer is a relative term since it depends on thenature, strength and concentration of the acid or base added to orgenerated in the solution of interest. A weak buffer is a buffer thatallows detection (and therefore is not able to control or mask) pHchanges on the order of those listed above.

The reaction solution may have a buffer concentration equal to or lessthan 1 mM, equal to or less than 0.9 mM, equal to or less than 0.8 mM,equal to or less than 0.7 mM, equal to or less than 0.6 mM, equal to orless than 0.5 mM, equal to or less than 0.4 mM, equal to or less than0.3 mM, equal to or less than 0.2 mM, equal to or less than 0.1 mM, orless including zero. The buffer concentration may be 50-100 μM. Anon-limiting example of a weak buffer suitable for the sequencingreactions described herein wherein pH change is the readout is 0.1 mMTris or Tricine.

In some aspects, in addition to or instead of using reduced bufferingsolutions, nucleotide incorporation (and optionally excision) is carriedout in the presence of additional agents which serve to shield potentialbuffering events that may occur in solution. These agents are referredto herein as buffering inhibitors since they inhibit the ability ofcomponents within a solution or a solid support in contact with thesolution to sequester and/or otherwise interfere with released hydrogenions prior to their detection by the chemFET surface. In the absence ofsuch inhibitors, released hydrogen ions may interact with or besequestered by reactive groups in the solution or on solid supports incontact with the solution. These hydrogen ions are less likely to reachand be detected by the chemFET surface, leading to a weaker signal thanis otherwise possible. In the presence of such inhibitors however therewill be fewer reactive groups available for interaction with orsequestration of hydrogen ions. As a result, a greater proportion ofreleased hydrogen ions will reach and be detected by the chemFETsurface, leading to stronger signals. Reactive groups that can interferewith released hydrogen ions include without limitation reactive groupssuch as free bases on single stranded nucleic acids and Si—OH groupsthat may be present in the passivation layer. Some suitable bufferinginhibitors demonstrate little or no buffering capacity in the pH rangeof 5-9, meaning that pH changes on the order of 0.005, 0.01, 0.02, 0.03,0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 or more pH units are detectable(e.g., by using an ISFET) in the presence of such inhibitors:

There are various types of buffering inhibitors. One example of abuffering agent is an agent that binds to single stranded nucleic acids(or single stranded nucleic acid regions, as may occur in a templatenucleic acid) thereby shielding reactive groups such as free bases.These agents may be RNA oligonucleotides (or RNA oligomers, oroligoribonucleotides, as they are referred to herein interchangeably)having complementary sequences to the afore-mentioned single strandedregions of template nucleic acids. RNA oligonucleotides are usefulbecause they are not able to serve as primers for a sequencing reactionas compared to DNA oligonucleotides. In order to bind to (or shield theeffects of) as much of a single stranded nucleic acid as possible, aplurality (or set, or mixture) of RNA oligonucleotides can be used. Asan example, a set of RNA oligonucleotides that are 2, 3, 4, 5, 6, ormore nucleotides in length can be used together with single strandedtemplates. The short length of these RNA oligonucleotides allows them tobe displaced by the polymerase as it progresses along with the length ofthe nucleic acid template. Such displacement does not requireexonuclease activity from the polymerase. Typically, the RNAoligonucleotides are of random sequence. In some embodiments, this ispreferred as no prior knowledge of the sequence of the single strandedregion of the template is required. FIGS. 61B and 61C illustrate thedifference in ion detection at an ISFET in the presence or absence of aRNA hexamer bound to a single stranded template.

Another example of a class of buffering inhibitors is phospholipids. Thephospholipids may be naturally occurring or non-naturally occurringphospholipids. Examples of phospholipids that may be used as bufferinginhibitors include but are not limited to phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine.

Another example of a buffering inhibitor is sulfonic acid basedsurfactants such as poly(ethylene glycol) 4-nonylphenyl 3-sulfopropylether (PNSE), the potassium salt of which is shown in FIG. 61D. Inaddition to shielding reactive groups that would otherwise. interferewith released protons, PNSE has also been reported to enhance polymeraseactivity.

Another example of a buffering inhibitor is polyanionic electrolytessuch as poly(styrenesulfonic acid), the sodium salt of which is shown inFIG. 61E.

Another example of a buffering inhibitor is polycationic electrolytessuch as poly(diallydimethylammonium), the chloride salt of which isshown in FIG. 61F. These compounds are known to bind to DNA.

Another example of a buffering inhibitor is tetramethyl ammonium, thechloride salt of which is shown in FIG. 61G.

These various inhibitors may be present throughout a reaction by beingincluded in nucleotide solutions, wash solutions, and the like.Alternatively, they may be flowed through the chamber at set timesrelative to the flow through of nucleotides and/or other reactionreagents. In still other embodiments, they may be coated on the chemFETsurface (or reaction chamber surface). Such coating may be covalent ornon-covalent.

Another way of reducing the buffering capacity in the reaction well isto covalently attach nucleic acids to capture beads, in embodiments inwhich capture beads are used. Such covalent attachment is in contrast tonon-covalent methods described herein that include for example biotin,streptavidin interactions. In these latter embodiments, biotinylatedprimers can be attached to streptavidin coated beads, followed byhybridization to template. However, streptavidin, like other proteins,is capable of buffering, and therefore its presence would interfere withthe detection of hydrogen ions released as a consequence of nucleotideincorporation. Thus, the invention also contemplates in some instancesapproaches that do not rely on streptavidin in the attachment mechanism.One such alternative involves covalently coupling primers to beads(and/or other solid supports such as the chemFET surface). Covalentlycoupling primers to such solid supports serves at least two purposes.First, it eliminates the need for proteins, such as streptavidin, thatcomprise functional side groups (such as primary, secondary or tertiaryamines and carboxylic acids) that can buffer pH changes in the range ofpH 5-9. Second, it serves to increase the number of templates that canbe conjugated to the solid support, such as a single bead, by reducingsteric hindrance effects that may exist when using bulky proteins suchas streptavidin. In still other embodiments, templates may be directlyconjugated covalently to solid supports such as beads.

Primers can be covalently coupled to beads in any number of ways,several of which are shown in FIGS. 61H and 61I or described inSteinberg et al. Biopolymers 73:597-605, 2004, as an example. Reactivegroups that can be used to conjugate primers to beads include epoxide,tosyl, amino and carboxyl groups. In addition, beads having a silicasurface, as discussed below, can be used with chlorophyl, azide, andalkyne reactive groups. In some embodiments, the preferred combinationis a polymer core bead with a polymer surface using tosyl reactivegroups.

Increasing the number of templates or primers (i.e., copy number)results in a greater number of nucleotide incorporations per sensorand/or per reaction chamber, thereby leading to a higher signal and thussignal to noise ratio. Copy number can be increased for example by usingtemplates that are concatemers (i.e., nucleic acids comprising multiple,tandemly arranged, copies of the nucleic acid to be sequenced), byincreasing the number of nucleic acids on or in beads up to andincluding saturating such beads, and by attaching templates or primersto beads or to the sensor surface in ways that reduce steric hindranceand/or ensure template attachment (e.g., by covalently attachingtemplates), among other things. Concatemer templates may be immobilizedon or in beads or on other solid supports such as the sensor surface,although in some embodiments concatemers templates may be present in areaction chamber without immobilization. For example, the templates (orcomplexes comprising templates and primers) may be covalently ornon-covalently attached to the chemFET surface and their sequencing mayinvolve detection of released hydrogen ions and/or addition of negativecharge to the chemFET surface upon a nucleotide incorporation event. Thelatter detection scheme may be performed in a buffered environment orsolution (i.e., any changes in pH will not be detected by the chemFETand thus such changes will not interfere with detection of negativecharge addition to the chemFET surface).

For some aspects described herein, it is important that bufferingcapacity not be affected in the process of increasing copy number. Thus,various methods are provided for increasing copy number using strategiesand/or linkers that do not impact the buffering capacity of theenvironment. In some instances, the functional groups, linkers and/orpolymers themselves have no or limited buffering capacity, and their usedoes not obscure the detection of hydrogen ions released as a result ofnucleotide incorporation or excision, as the case may be.

Increasing copy number may also be accomplished by increasing the numberof attachment points for primers (or templates). Some of thesemethodologies are described below.

In one embodiment, the solid support is coated with a polymer such aspolyethylene glycol (PEG) which does not comprise functional groups thatinteract with the primer and its functional groups, except as providedbelow for initially attaching primer. PEG linkers of varying lengths canbe used so that primers can be attached at varying distances from thesolid support surface, thereby decreasing the amount of steric hindrancethat may otherwise exist between primers and the complexes theyultimately form (e.g., primer/template hybrids). The solid supports canbe coated one or more times with a mixture of 2, 3, 4, or more PEGlinkers of differing lengths. The end result is an increased distancebetween ends of PEG linkers attached to the solid support. Attachment ofprimers to the PEG linkers can be accomplished using any reactive groupsknown in the art. As an example, click chemistry can be used betweenazide groups on the ends of PEG linkers and alkyde groups on theprimers.

In another embodiment, polymers having preferably more than onefunctional (or reactive) group are used. Each of the functional groupsis available for conjugation with a separate primer. Useful polymers inthis regard include those having hydroxyl groups, amine groups, thiolgroups, and the like. Examples of suitable polymers include dextran andchitosan. Linear or branched forms of these polymers may be used. Anexample of a branched polymer with multiple functionalities is brancheddextran. It will be apparent to those of ordinary skill in the art thanany chimeric polymer or copolymer may also be used provided it has asufficient number of functional groups for primer attachment.

Yet another embodiment involves the use of dendrimers and preferablyhigher order dendrimers to bind primer. Dendrimers are three-dimensionalcomplexes that can be made having any functional group. Examples ofdendrimers include the PAMAM dendrimers, an example of which is CAS No.163442-69-1 which has 256 amine groups. Dendrimers are commerciallyavailable from sources such as Sigma-Aldrich and DendriticNanotechnologies Inc. It will be understood that dendrimers with otherfunctional groups also can be used.

The invention further contemplates the use of any combination of theabove embodiments for maximizing the number of primers attached to asolid support. Thus for example the solid support surface may be coatedone or more times (e.g., once or twice) with the PEG linkers of varyinglengths, and to such linkers may be attached multifunctionality polymerssuch as dextran or chitosan (in either linear or branched form),followed by attachment of primers. As another example, dendrimers may beattached to the PEG linkers, followed by primer attachment to thedendrimers.

In still another embodiment, the invention contemplates coating thesolid support surface with a population of self-assembling monomers someproportion of which are bound to primers. As an example, the monomersmay be acrylamide monomers some of which are attached to primers. Theend result is a solid support having a polyacrylamide coating withinterspersed primers. The density of primers bound to the solid supportcan be manipulated by changing the ratio of monomers that have primersand monomers that lack primers. This strategy has been reported byRehman et al. Nucleic Acids Research, 1999, 27(2):649-655.

Still other methods for attaching nucleic acids to beads are taught byLund et al., Nucleic Acids Research, 1988, 16(22):10861-10880, Joos etal. Anal Biochem, 1997, 247:96-101, Steinberg et al. Biopolymers, 2004,73:597-605, and Steinberg-Tatman et al. Bioconjugate Chem 200617:841-848.

Beads can be made of any material including but not limited tocellulose, cellulose derivatives, gelatin, acrylic resins, glass, silicagels, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide,polystyrene, polystyrene cross-linked with divinylbenzene or the like(see, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamides,latex gels, dextran, crosslinked dextrans (e.g., Sephadex™), rubber,silicon, plastics, nitrocellulose, natural sponges, metal, and agarosegel (Sepharose™). In one embodiment, the beads are streptavidin-coatedbeads.

Beads suitable for covalent attachment may be magnetic or non-magneticin nature. They may have a polymer core with a polymer surface, apolymer core with a silica surface, and a silica core with a silicasurface. The bead core may be hollow, porous, or solid, as describedbelow.

The bead diameter will depend on the density of the chemFET andmicrowell arrays used, with larger arrays (and thus smaller sized wells)requiring smaller beads. Generally the bead size may be about 1-10microns, and more preferably 2-6 microns. In some embodiments, the beadsare about 5.9 microns while in other embodiments the beads are about 2.8microns. In still other embodiments, the beads are about 1.5 microns, orabout 1 micron in diameter. In some embodiments, beads having a diameterthat ranges from about 3.3 to 3.5 microns may be used for reaction wellarrays having a pitch on the order of about 5.1 microns. In otherembodiments, beads having a diameter that ranges from about 5 to 6.5microns may be used for reaction well arrays having a pitch on the orderto about 9 microns. It is to be understood that the beads may or may notbe perfectly spherical in shape. It is also to be understood that otherbeads may be used and other mechanisms for attaching the nucleic acid tothe beads may be used. In some instances the capture beads (i.e., thebeads on which the sequencing reaction occurs) are the same as thetemplate preparation beads including the amplification beads. In someinstances, even where non-covalent attachment is contemplated, a spaceris used to distance the template nucleic acid (and in particular thetarget nucleic acid sequence comprised therein) from a solid supportsuch as a bead. This facilitates sequencing of the end of the targetclosest to the bead, for instance. Examples of suitable linkers areknown in the art (see Diehl et al. Nature Methods, 2006, 3(7):551-559)and include but are not limited to carbon-carbon linkers such as but notlimited to iSpl8. Beads can be purchased from commercial suppliers suchas Bangs, Dynal and Micromod. Additional spacers and nucleic acidattachment mechanisms are discussed above.

As stated above, some beads may be solid while others may be porous orhollow. These beads will have a porous surface such that reagents fromthe reaction solution may move into and out of the bead These may haveempty channels or hollow cores that comprise at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% of the bead volume. These beads will be referredto herein as porous beads, porous microparticles, or capsules in view oftheir non-solid cores, and these terms are intended to embrace porous aswell as hollow beads regardless of diameter or volume. They may or maynot be spherical.

The invention contemplates the use of such porous beads in the varioussequencing methods described herein. More specifically, the inventioncontemplates sequencing nucleic acids that are present in porous beads.Porous beads may be generated by methods known in the art. See forexample Mak et al. Adv. Funct. Mater. 2008 18:2930-2937; Morimoto et al.MEMS 2008 Tucson Ariz. USA Jan. 13-17, 2008 Poster Abstract 304-307; Leeet al. Adv. Mater. 2008 20:3498-3503; Martin-Banderas et al. Small. 20051(7):688-92; and published PCT application WO03/078659.

Porous microparticles may be initially generated to contain a singletemplate nucleic acid which is later amplified with all amplified copiesof the nucleic acid being retained in the microparticle. Amplificationmay occur before or while the bead is in contact with a chemFET array,and/or optionally in a reaction chamber. If performed before contactwith the chemFET array, beads that have successfully undergoneamplification can be selected and thereby enriched. As an example, beadshaving amplified nucleic acids can be separated from other beads basedon density. Amplification may be isothermal or PCR amplification, orother means of amplification, as the invention is not to be limited inthis regard. The beads may contain at least two types of enzymes such astwo types of polymerases. For example, the beads may contain one type ofpolymerase that is suitable for amplification of the nucleic acid and asecond type of polymerase that is suitable for sequencing the amplifiednucleic acids. The beads preferably contain a plurality of both types ofpolymerases and preferably the number of each polymerase will be inexcess of a saturating amount so as not to create a polymerase-limitedenvironment. Once amplification is completed, the amplificationpolymerase may be inactivated, while maintaining the activity of thesequencing polymerase. Typically, the enzymes and nucleic acids will beretained in the bead while smaller compounds, such as dNTPs and othernucleic acid synthesis reagents and cofactors, are allowed to diffuseinto and out of the bead. Importantly for the invention, synthesisbyproducts such as PPi and hydrogen ions will also diffuse out of thebeads, in order to be detected by the chemFET.

Nick Translation

The invention provides, in various other aspects, other modes foranalyzing, including for example sequencing, nucleic acids usingreactions that involve interdependent nucleotide incorporation andnucleotide excision. As used herein, interdependent nucleotideincorporation and nucleotide excision means that both reactions occur onthe same nucleic molecule at contiguous sites on the nucleic acid, andone reaction facilitates the other.

An example of such a reaction is a nick translation reaction. A nicktranslation reaction, as used herein, refers to a reaction catalyzed bya polymerase enzyme having 5′ to 3′ exonuclease activity, that involvesincorporation of a nucleotide onto the free 3′ end of a nicked region ofdouble stranded DNA and excision of a nucleotide located at the free 5′end of the nicked region of the double stranded DNA. Nick translationtherefore refers to the movement of the nicked site along the length ofthe nicked strand of DNA in a 5′ to 3′ direction. As will be recognizedby those of ordinary skill in the art, the nick translation reactionincludes a sequencing-by-synthesis reaction based on the intact strandof the double stranded DNA. This strand acts as the template from whichthe new strand is synthesized. The method does not require the use of aprimer because the double stranded DNA can prime the reactionindependently. These aspects of the invention will refer specifically tonick translation for the sake of brevity, but it is to be understoodthat any other combined reaction of nucleotide excision andincorporation will be equally and fully intended in the followingdiscussion.

The nick translation approach has two features that make it well suitedto the detection methods provided herein. First, the nick translationreaction results in the release of two hydrogen ions for each combinedexcision/incorporation step, thereby providing a more robust signal atthe chemFET each time a nucleotide is incorporated into a newlysynthesized strand. A sequencing-by-synthesis method, in the absence ofnucleotide excision, releases one hydrogen ion per nucleotideincorporation. In contrast, nick translation releases a first hydrogenion upon incorporation of a nucleotide and a second hydrogen ion uponexcision of another nucleotide. This increases the signal that can besensed at the chemFET, thereby increasing signal to noise ratio andproviding a more definitive readout of nucleotide incorporation.

Second, the use of a double stranded DNA template (rather than a singlestranded DNA template) results in less interference of the template withreleased ions and a better signal at the chemFET. A single stranded DNAhas exposed groups that are able to interfere with (for example,sequester) hydrogen ions. These reactive groups are shielded in a doublestranded DNA where they are hydrogen bonded to complementary groups. Bybeing so shielded, these groups do not substantially impact hydrogen ionlevel or concentration. As a result, signal resulting from hydrogen ionrelease is greater in the presence of double stranded as compared tosingle stranded templates, as will be signal to noise ratio, therebyfurther contributing to a more definitive readout of nucleotideincorporation.

Templates suitable for nick translation typically are completely orpartially double stranded. Such templates comprise an opening (or anick) which acts as an entry point for a polymerase. Such openings canbe introduced into the template in a controlled manner as describedbelow and known in the art.

As will be appreciated by one of ordinary skill in the art, it ispreferable that these openings be present in each of the plurality ofidentical templates at the same location in the template sequence.Typical molecular biology techniques involving nick translation userandomly created nicks along the double stranded DNA because their aimis to produce a detectably labeled nucleic acid. These prior art methodsgenerate nicks through the use of sequence-independent nicking enzymessuch as DNase I. In the methods of the invention however the nicklocation must be known, non-random and uniform for all templates ofidentical sequence. There are various ways of achieving this, and someof these are discussed below.

One way of achieving this is to create a population of identical doublestranded nucleic acid templates that comprise a uracil residue in adefined location on one strand. The uracil may be present in a primerthat is used to generate the double stranded nucleic acid or a probethat is hybridized to a single stranded region of a predominantly doublestranded nucleic acid. The population of identical template nucleicacids can be generated by an amplification reaction, for example a PCRreaction. The PCR reaction can be performed using a primer pair, one ofwhich comprises a uracil residue. Alternatively, the PCR reaction can beperformed with non-uracil containing primers, followed by denaturationof the double stranded amplified products, and hybridization of onestrand to a uracil-containing primer. This latter embodiment requiresthat the single stranded, primed templates be made double stranded priorto the nick translation reaction. These reactions may be carried outwhile the nucleic acids are bound to a solid support such as a bead.Alternatively, the double stranded nucleic acid templates may be firstgenerated and then attached to a solid support.

The uracil-containing double stranded nucleic acids are then contactedwith uracil DNA glycosylase (UDG). UDG is an enzyme that removes uracilfrom DNA by cleaving the N-glycosylic bond. In some instances, thenucleic acid is contacted with a second enzyme that removes uracil. Thesecond enzyme may be an AP endonuclease, or a lyase or another enzymehaving similar nuclease activity. The nucleic acids may be in thereaction chamber (or well) discussed herein during exposure to theseenzymes, or they may be added to the reaction chamber (or well)following enzyme contact. Following contact with one (in some instances)or both enzymes, the double stranded nucleic acid comprises a nick at aspecific location. More importantly, all nucleic acids of the samesequence and treated in an identical manner will be nicked at the samelocation. These nicked nucleic acids can then be used as templates fornucleic acid sequencing or other analysis.

Another way in which double stranded nucleic acids can be uniformlynicked is similar to the method just described with the exception that anucleotide sequence recognized by a nickase or nicking enzyme isincorporated into the nucleic acid. The nickase cuts on only one strandof the double stranded DNA. Some nickases cut their recognition sequencewhile others cut at a distance from their recognition sequence (e.g.,type II nickases). Nickases with longer recognition sites are preferredbecause such sites are more infrequent and thus less likely to bepresent in the target nucleic acid (e.g., the genomic fragment) includedin the template nucleic acid. Examples of single stranded sequencespecific nucleases (and their respective sequences) include withoutlimitation Nb.BbvCI (CCTCA↓GC), Nt.BbvCI (CC↓TCAGC), Nb.BsmI (GAATG↓C),Nt.SapI (GCTCTTCN↓), Nb.BsrDI (GCAATG↓), and Nb.BtsI (GCAGTG↓), whereinthe arrow indicates the site of nicking. Nickases are commerciallyavailable from a number of suppliers including NEB. Accordingly, thenucleic acids are prepared having a copy of the nickase recognitionsequence in a region of known sequence (e.g., a primer or otherartificial sequence in the template nucleic acid). These nucleic acidsare then contacted with the corresponding nickase to nick the nucleicacid. As with the uracil embodiment, contact with the nickase can occurbefore or after the nucleic acids are attached to solid support such asbeads, and before or after the nucleic acids are loaded in reactionwells.

Still another way in which double stranded nucleic acids may beuniformly nicked is by incorporating ribonucleotides (rather thandeoxyribonucleotides) into one strand of the double stranded nucleicacids. This can be accomplished in a manner similar to that describedfor the generation of uracil-containing nucleic acids. In other words, adouble stranded nucleic acid can be generated using primers that containone or more ribonucleotides at predetermined and thus known positions.The resultant nucleic acids are then contacted with RNase H or otherenzyme that degrades the RNA portion of DNA-RNA hybrids. RNase H inparticular hydrolyses phosphodiester bonds of RNA in RNA:DNAheteroduplexes, thereby producing 3′ OH groups and 5′ phosphate groups.If the double stranded nucleic acid is generated with only a singleribonucleotide then only a single abasic site will result, whereas ifthe double stranded nucleic acid is generated with multipleribonucleotides then multiple abasic sites will result. In either case,identical nucleic acids can still be analyzed using a nick translationreaction once all but one of the abasic sites are filled by thepolymerase. Taq polymerase is preferred in some embodiments involvingthese RNA-DNA hybrids. Again, as with the other methods described above,contact with RNase H or other similar enzyme can occur before or afterthe nucleic acids are attached to a solid support such as beads, andbefore or after the nucleic acids are loaded in reaction wells.

Still another way to prepare double stranded nucleic acids suitable astemplates for nucleotide incorporation and excision events is togenerate a double stranded nucleic acid having a 3′ overhang on one end,and then subsequently hybridize to the 3′ overhang a nucleic acid thatis shorter than the overhang by at least one nucleotide. Preferably,after hybridization of the two nucleic acids to each other, there willbe one unpaired internal nucleotide in the overhang and this will be thesite from which nick translation will begin. Again, the sequence of the3′ overhang and the hybridizing nucleic acid will be known and thereforethe location of the abasic site will also be known and will be identicalfor all template nucleic acids. The hybridization can occur before orafter the nucleic acids are attached to a solid support such as beads,and before or after the nucleic acids are loaded into reaction wells.

Another example of a suitable nick translation template is aself-priming nucleic acid. The self priming nucleic acid may comprise adouble stranded and a single stranded region that is capable ofself-annealing in order to prime a nucleic acid synthesis reaction. Thesingle stranded region is typically a known synthetic sequence ligatedto a nucleic acid of interest. Its length can be predetermined andengineered to create an opening following self-annealing, and suchopening can act as an entry point for a polymerase.

It is to be understood that, as the term is used herein, a nickednucleic acid, such as a nicked double stranded nucleic acid, is anucleic acid having an opening (e.g., a break in its backbone, or havingabasic sites, etc.) from which a polymerase can incorporate andoptionally excise nucleotides. The term is not limited to nucleic acidsthat have been acted upon by an enzyme such as a nicking enzyme, nor isit limited simply to breaks in a nucleic acid backbone, as will be clearbased on the exemplary methods described herein for creating suchnucleic acids.

Once the nicked double stranded nucleic acids are generated, they arethen subjected to a nick translation reaction. If the nick translationreaction is performed to sequence the template nucleic acid, the nicktranslation can be carried out in a manner that parallels thesequencing-by-synthesis methods described herein. More specifically, insome embodiments each of the four nucleotides is separately contactedwith the nicked templates in the presence of a polymerase having 5′ to3′ exonuclease activity. In other embodiments, known combinations ofnucleotides are used. Examples of suitable enzymes include DNApolymerase I from E. coli, Bst DNA polymerase, and Taq DNA polymerase.The order of the nucleotides is not important as long as it is known andpreferably remains the same throughout a run. After each nucleotide iscontacted with the nicked templates, it is washed out followed by theintroduction of another nucleotide, just as described herein. In thenick translation embodiments, the wash will also carry the excisednucleotide away from the chemFET.

It should be appreciated that just as with other aspects and embodimentsdescribed herein the nucleotides that are incorporated into the nickedregion need not be extrinsically labeled since it is a byproduct oftheir incorporation that is detected as a readout rather than theincorporated nucleotide itself. Thus, the nick translation methods maybe referred to as label-free methods, or fluorescence-free methods,since incorporation detection is not dependent on an extrinsic label onthe incorporated nucleotide. The nucleotides are typically naturallyoccurring nucleotides. It should also be recognized that since themethods benefit from the consecutive incorporation of as manynucleotides as possible, the nucleotides are not for example modifiedversions that lead to premature chain termination, such as those used insome sequencing methods.

Target and Template Nucleic Acids

The nucleic acid being sequenced is referred to herein as the targetnucleic acid. Target nucleic acids include but are not limited to DNAsuch as but not limited to genomic DNA, mitochondrial DNA, cDNA and thelike, and RNA such as but not limited to mRNA, miRNA, and otherinterfering RNA species, and the like. The nucleic acids may benaturally or non-naturally occurring. They may be obtained from anysource including naturally occurring sources such as any bodily fluid ortissue that contains DNA, including, but not limited to, blood, saliva,cerebrospinal fluid (“CSF”), skin, hair, urine, stool, and mucus, orsynthetic sources. The nucleic acids may be PCR products, cosmids,plasmids, naturally occurring or synthetic libraries, and the like. Theinvention is not intended to be limited in this regard. It shouldtherefore be understood that the invention contemplates analysis,including sequencing, of DNA as well as RNA.

With respect to RNA, amplification methods such as the SMART system andNASBA are known in the art and have been reported by van Gelder et al.PNAS, 1990, 87:1663-1667, Chadwick et al. BioTechniques, 1998,25:818-822, Brink et al. J Clin Microbiol, 1998, 36(11):3164-3169,Voisset et al. BioTechniques, 2000, 29:236-240, and Zhu et al.BioTechniques, 2001, 30:892-897. The amplification methods described inthese references are incorporated by reference herein.

The starting amounts of nucleic acids to be sequenced determine theminimum sample requirements. Considering the following bead sizes, withan average of 450 bases in the single stranded region of a template,with an average molecular weight of 325 g/mol per base, Table 2 showsthe following:

TABLE 2 Bead Size (um) femto gram of DNA 0.2 0.124 0.3 0.279 0.7 1.521.05 3.42 2.8 24.3 5.9 108

Given the number of beads and microwells contemplated for use in anarray, in some embodiments of the invention, it will be apparent that asample taken from a subject to be tested need only be on the order of 3μg. Thus, the systems and methods described herein can be utilized tosequence an entire genome of an organism from about 3 μg of DNA or less.As discussed herein, such sequences can be obtained without the use ofoptics or extrinsic labels.

Target nucleic acids are prepared using any manner known in the art. Asan example, genomic DNA may be harvested from a sample according totechniques known in the art (see for example Sambrook et al.“Maniatis”). Following harvest, the DNA may be fragmented to yieldnucleic acids of smaller length. The resulting fragments may be on theorder of hundreds, thousands, or tens of thousands nucleotides inlength. In some embodiments, the fragments are 200-1000 base pairs insize, or 300-800 base pairs in size, about 200, about 300, about 400,about 500, about 600, about 700, about 800, about 900, or about 1000base pairs in length, although they are not so limited.

Nucleic acids may be fragmented by any means including but not limitedto mechanical, enzymatic or chemical means. Examples include shearing,sonication, nebulization, endonuclease (e.g., DNase I) digestion,amplification such as PCR amplification, or any other technique known inthe art to produce nucleic acid fragments, preferably of a desiredlength. As used herein, fragmentation also embraces the use ofamplification to generate a population of smaller sized fragments of thetarget nucleic acid. That is, the target nucleic acids may be melted andthen annealed to two (and preferably more) amplification primers andthen amplified using for example a thermostable polymerase (such asTaq). An example is a massively parallel PCR-based amplification.Fragmentation can be followed by size selection techniques to enrich orisolate fragments of a particular length or size. Such techniques arealso known in the art and include but are not limited to gelelectrophoresis or SPRI.

Alternatively, target nucleic acids that are already of sufficientlysmall size (or length) may be used. Such target nucleic acids includethose derived from an exon enrichment process. Thus, rather thanfragmenting (randomly or non-randomly) longer target nucleic acids, thetargets may be nucleic acids that naturally exist or can be isolated inshorter, useable lengths such as mRNAs, cDNAs, exons, PCR products (asdescribed above), and the like. See Albert et al. Nature Methods 20074(11):903-905 (microarray hybridization of exons and locus-specificregions), Porreca et al. Nature Methods 2007 4(11):931-936, and Okou etal. Nature Methods 2007 4(11):907-909 for methods of isolating and/orenriching sequences such as exons prior to sequencing.

The target nucleic acids are typically ligated to adaptor sequences onboth the 5′ and 3′ ends. The resulting nucleic acid is referred toherein as a template nucleic acid. The template nucleic acid thereforecomprises at least the target nucleic acid and usually comprisesnucleotide sequences in addition to the target at both the 5′ and 3′ends. The template nucleic acids may be engineered such that differenttemplates have identical 5′ ends and identical 3′ ends. The 5′ and 3′ends in each individual template are preferably different in sequence.

Adaptor sequences may comprise sequences complementary to amplificationprimer sequences, to be used in amplifying the target nucleic acids. Oneadaptor sequence may also comprise a sequence complementary to thesequencing primer (i.e., the primer from which sequencing occurs). Theopposite adaptor sequence may comprise a moiety that facilitates bindingof the nucleic acid to a solid support such as but not limited to abead. An example of such a moiety is a biotin molecule (or a doublebiotin moiety, as described by Diehl et al. Nature Methods, 2006,3(7):551-559) and such a labeled nucleic acid can therefore be bound toa solid support having avidin or streptavidin groups. Another moietythat can be used is the NHS-ester and amine affinity pair. It is to beunderstood that the invention is not limited in this regard and one ofordinary skill is able to substitute these affinity pairs with otherbinding pairs. In some embodiments, the solid support is a bead and inothers it is a wall of the reaction chamber (or well) such as a bottomwall or a side wall, or both.

In some embodiments, the invention contemplates the use of a pluralityof template populations, wherein each member of a given plurality sharesthe same 3′ end but different template populations differ from eachother based on their 3′ end sequences. As an example, the inventioncontemplates in some instances sequencing nucleic acids from more thanone subject or source. Nucleic acids from a first source may have afirst 3′ sequence, nucleic acids from a second source may have a second3′ sequence, and so on, provided that the first, second, and anyadditional 3′ sequences are different from each other. In this respect,the 3′ end, which is typically a unique sequence, can be used as abarcode or identifier to label (or identify) the source of theparticular nucleic acid in a given well. Reference can be made to Meyeret al. Nucleic Acids Research 2007 35(15):e97 for a discussion oflabeling nucleic acid with barcodes followed by sequencing.

Templates disposed onto a chemFET array (and thus over more than onesensor in the array) may share identical primer binding sequences. Thisfacilitates the use of an identical primer across microwells and alsoensures that a similar (or identical) degree of primer hybridizationoccurs across microwells. Once annealed to complementary primers such assequencing primers, the templates are in a complex referred to herein asa template/primer hybrid. In this hybrid, at least one region of thetemplate is double stranded (i.e., where it is bound to itscomplementary primer) and in some instances the remaining region of thetemplate is single stranded. It is this single stranded region that actsas the template for the incorporation of nucleotides to the end of theprimer and thus it is also this single stranded region which isultimately sequenced according to the invention. As discussed herein,this single stranded region may be bound by short RNA oligomers, ofknown or unknown (i.e., random) sequence, and still capable of beingsequenced.

In some embodiments, the template nucleic acid is able to self-annealthereby creating a 3′ end from which to incorporate nucleotidetriphosphates. Thus in such instances, there is no need for a separatesequencing primer since the template acts as both template and primer.See Eriksson et al. Electrophoresis 25:20-27, 2004 for a discussion ofthe use of self-annealing template in a pyrosequencing reaction. Inother instances, sequencing primers are hybridized (or annealed, as theterms are used interchangeably herein) to the templates prior tointroduction or contact with the chemFET or reaction chamber.

The plurality of templates in each microwell may be introduced into themicrowells (e.g., via a nucleic acid loaded bead), or it may begenerated in the microwell itself. A plurality is defined herein as atleast two, and in the context of template nucleic acids in a microwellor on a nucleic acid loaded bead includes tens, hundreds, thousands, tenthousands, hundred thousands, millions, or more copies of the templatenucleic acid. The limit on the number of copies will depend on a numberof variables including the number of binding sites for template nucleicacids (e.g., on the beads or on the walls of the microwells), the sizeof the beads, the length of the template nucleic acid, the extent of theamplification reaction used to generate the plurality, and the like. Itis generally preferred to have as many copies of a given template perwell in order to increase signal to noise ratio as much as possible, asdiscussed herein. In some embodiments, the amplification is arepresentative amplification. A representative amplification is anamplification that does not alter the relative representation of anynucleic acid species.

Thus, the template nucleic acid may be amplified prior to or afterplacement in the well and/or contact with the sensor. Amplification andconjugation of nucleic acids to solid supports such as beads may beaccomplished in a number of ways. For example, in one aspect once atemplate nucleic acid is loaded into a well of the flow cell 200,amplification may be performed in the well, the resulting amplifiedproduct denatured, and sequencing-by-synthesis then performed. In oneembodiment, the template is amplified in solution and then hybridized toa single primer that is immobilized on the chemFET surface. The use ofonly one primer type on the surface ensures that only one of theamplified strands is eventually bound to the surface, and the otherstrand is removed through wash.

Amplification methods include but are not limited to emulsion PCR (i.e.,water in oil emulsion amplification) as described by Margulies et al.Nature 2005 437(15):376-380 and accompanying supplemental materials,bridge amplification, rolling circle amplification (RCA), concatemerchain reaction (CCR), or other strategies using isothermal ornon-isothermal amplification techniques.

Bridge amplification can be used to produce a solid support (such as areaction chamber wall or a bead) having amplified copies of the sametemplate. The method involves contacting template nucleic acids with thechemFET/reaction chamber array at a limiting dilution in order to ensurethat reaction chambers contain only a single template. The chemFETsurface will typically be coated with two populations of primers. In oneembodiment, the chemFET surface is coated with both forward and reverseprimers that are complementary to the engineered 5′ and 3′ sequences ofthe template. The template is bound to the chemFET surface directly andthen allowed to hybridize at its free end with a complementary primer onthe surface. The primer is extended using unlabeled nucleotides, and theresultant double stranded nucleic acid is then denatured. This resultsin immobilized copies of the template nucleic acid and its complement inclose proximity on the surface. This process is repeated by allowing thetemplate and its complement to hybridize at their free ends to otherprimers on the surface. The net result is a population of immobilizedtemplate and a population of immobilized complement that areinterspersed amongst each other. The sequencing-by-synthesis reaction isthen carried out using a sequencing primer that binds to one but notboth immobilized strands. This effectively selects for one of thestrands and ensures that only one strand is sequenced. Either strand canbe sequenced since they are complements of each other.

In a related embodiment, the solid support is a bead and the bead iscoated with the two primer populations and only a single strandedtemplate nucleic acid, at least initially. This amplification method isdescribed in U.S. Pat. No. 5,641,658 to Adams et al.

In still another embodiment, each solid support surface (whether bead orreaction chamber wall) has bound thereto a specific and unique primerpair that may be but is not limited to a gene specific primer pair. Oneor both of the primers in the pair select for templates in a librarythat is applied to the solid support. Due to the unique sequence of theprimers, it is expected that only the desired template will hybridizeand then be amplified and sequenced, as described above.

RCA or CCR amplification methods generate concatemers of templatenucleic acids that comprise tens, hundreds, thousands or more tandemlyarranged copies of the template. Such concatemers may still be referredto herein as template nucleic acids, although they may contain multiplecopies of starting template nucleic acids. In some embodiments, they mayalso be referred to as amplified template nucleic acids. Alternatively,they may be referred to herein as comprising multiple copies of targetnucleic acid fragment. Concatemers may contain 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or more copies ofthe starting nucleic acid. They may contain 10-10², 10²-10³, 10³-10⁴,10³-10 ⁵, or more copies of the starting nucleic acid. Concatemersgenerated using these or other methods (such as for example DNAnanoballs) can be used in the sequencing-by-synthesis methods describedherein. The concatemers may be generated in vitro apart from the arrayand then placed into reaction chambers of the array or they may begenerated in the reaction chambers. One or more inside walls of thereaction chamber may be treated to enhance attachment and retention ofthe concatemers, although this is not required. In some embodiments ofthe invention, if the concatemers are attached to an inside wall of thereaction chamber, such as the chemFET surface, then nucleotideincorporation at least in the context of a sequencing-by-synthesisreaction may be detected by a change in charge at the chemFET surface,as an alternative to or in addition to the detection of releasedhydrogen ions as discussed herein. If the concatemers are deposited ontoa chemFET surface and/or into a reaction chamber,sequencing-by-synthesis can occur through detection of released hydrogenions as discussed herein. The invention embraces the use of otherapproaches for generating concatemerized templates. One such approach isa PCR described by Stemmer et al. in U.S. Pat. No. 5,834,252, and thedescription of this approach is incorporated by reference herein.

The ability to use template nucleic acids independently of beads andthat can be deposited into reaction chambers or onto chemFET surfacesfacilitates the use of dense chemFET arrays. As will be understood,denser arrays will typically incorporate more chemFETs and optionallymore reaction chambers (where they are used) per array (or chip). Inorder to accommodate the increased number of chemFETs and optionallyreaction chambers, the size of the chemFETs and optionally reactionchambers is reduced. Accordingly, in some instances, it may bepreferable to use nucleic acids that are concatemers of the nucleic acidto be sequenced, independently of beads. Such nucleic acids may beallowed to self-assemble onto a treated chemFET surface, or they maysettle into the well (for example, by gravity), or they may be pulled inby magnetic or other force. Thus, the invention contemplates the use ofsuch concatemerized template nucleic acids in the pH basedsequencing-by-synthesis methods described herein.

As discussed herein, one approach for generating nucleic acids thatcomprise multiple copies of a nucleic acid to be sequenced involvesamplification of a circular template. The resultant amplified productforms a three dimensional structure that may occupy a spherical volumeor other three dimensional volume and shape. The occupied volume mayvary, depending on the size of the resultant nucleic acid. For example,in some instances the spherical volume may have an average diameter onthe order of about 100-300 nm. The generation of these three dimensionalstructures is described further in published US patent applicationsUS20070072208A1 and US20070099208A1, both to Drmanac et al.

Such nucleic acids may be generated in solution (i.e., amplificationoccurs in solution) and therefore emulsion based techniques or reactionchambers or wells are not necessary in some instances. As each resultantnucleic acid consists of a clonal amplified population of a startingnucleic acid, there will be no cross contamination of nucleic acids andnor does there have to be any physical separation between individualamplification reactions. Thus, in some aspects, it is contemplated thatnucleic acids (such as “DNA nanoballs” or “amplicons”) are generated insolution and then deposited onto chemFET surfaces and/or into reactionchambers. Further references that describe amplifications methodssuitable for the synthesis of these nucleic acids include U.S. Pat. Nos.4,683,195, 4,965,188, 4,683,202, 4,800,159, 5,210,015, 6,174,670,5,399,491, 6,287,824, 5,854,033 and published US patent applicationUS20060024711. Linear rolling circle amplification, multipledisplacement amplification, and padlock probe rolling circleamplification can all be used to generate clonal amplicons without theneed for limiting dilution in order to avoid cross-contamination ofnucleic acid templates by each other.

The chemFET surfaces may be treated (or patterned) or untreated (orunpatterned). In some instances, treated (or patterned) surfaces arepreferred in order to maximize nucleic acid deposition and/or retentiononto a surface. It is further known in the art that these nucleic acidsmay self-assemble onto the chemFET surface provided the chemFET arraysurface comprises regions to which the nucleic acids bind and optionallyregions to which they do not bind. Additionally, the binding of anucleic acid to one region on the surface will repel the binding ofanother nucleic acid, thereby precluding the possibility that two ormore nucleic acids of different sequence could co-exist at the samechemFET surface. The chemFET array may have an occupancy on the order ofgreater than 50%, greater than 60%, greater than 70%, greater than 80%,or 90% or greater (i.e., the number of individual chemFET surfaces ontowhich a single nucleic acid is deposited). It will be understood that,as used herein, the term deposited refers simply to the placement of thenucleic acid in close proximity and potentially in contact with achemFET surface (and optionally reaction chamber), but it does notrequire any particular interaction, whether covalent or non-covalent,between the nucleic acid and the chemFET surface.

The amplified nucleic acids discussed herein may be attached to thechemFET surface through functionalities incorporated into (e.g., duringamplification) or added post-synthesis to the nucleic acid. Suchfunctionalities may be located at adaptor regions within the nucleicacid which are not intended for sequencing according to the methodsprovided herein. For example, a concatemer may be generated from acircular template having two or more adaptor sequences (or nucleicacids) located upstream and downstream of the nucleic acids beingsequenced. Alternatively, the starting (or initial) nucleic acid mayconsist of a single adaptor sequence and a single nucleic acid to besequenced and in the process of amplification (such as, for example,RCA) the adaptor sequence is used to separate the copies of the nucleicacid to be sequenced from each other. Whether in this embodiment orothers described herein, functionalities present in the adaptorsequences may be used to attach and/or retain the resultant amplifiednucleic acids on a chemFET surface and optionally a reaction chamber.Exemplary functionalities include but are not limited to amino groups,sulfhydryl groups, carbonyl groups, biotin, streptavidin, avidin, amineallyl labeled nucleotides, NHS-ester interaction, thioether linkages,and the like.

Attachment may be via non-covalent bonds between capture nucleic acidspresent on the chemFET surface and complementary sequences in theadapter regions, or adsorption to the surface via Van der Waals forces,hydrogen bonding, static charge interactions, ionic and hydrophobicinteractions, and the like. Techniques used to attach DNAs tomicroarrays may also be used to attach the amplified products to thechemFET surface. These techniques include but are not limited to thosedescribed by Smirnov, Genes, Chrom & Cancer 40:72-77, 2004 and BeaucageCurr Med Chem 8:1213-1244, 2001.

Deposition and/or retention may also be accomplished using magneticforces. In these embodiments, magnetic particles may be incorporatedinto and/or attached post-synthesis to the amplified nucleic acids(e.g., at regions not intended for sequencing). Once the nucleic acidsare distributed on a chemFET array and optionally a reaction chamberarray, the array is placed in proximity to a magnet in order to move thenucleic acids towards the chemFET surface and optionally into a reactionchamber.

It should also be understood that the methods described hereincontemplate the synthesis of the amplified nucleic acids on or inproximity to the chemFET and optionally in a reaction chamber inaddition to synthesis in solution followed by deposition onto thechemFET surface. It is expected however that the latter approach willresult in a greater degree of occupancy of chemFET surfaces in thearray.

Accordingly, provided herein is an array of nucleic acids comprising aplurality of chemFETs each having a surface, and a plurality of nucleicacids, each nucleic acid deposited onto (or attached to) individualchemFET surfaces, wherein each nucleic acid comprises multiple identicalcopies of an initial nucleic acid to be sequenced. In some instances,the nucleic acid has a random coil state.

Also provided herein is a method for sequencing a nucleic acid presentin a reaction chamber of a reaction chamber array, comprisingsynthesizing a concatemer of a starting nucleic acid, wherein theconcatemer has a cross-sectional diameter greater than the diameter ofthe reaction well, optionally immobilizing (whether covalently ornon-covalently) the concatemer in the reaction chamber, and sequencingthe concatemer, preferably by sequencing-by-synthesis methods providedherein (e.g., pH based sequencing-by-synthesis methods). It will beunderstood that if the reaction chamber has a non-circular cross-sectionthen one or more or an average of cross-sectional dimensions can be used(as can a cross-sectional area) in comparing the concatemer and thereaction chamber sizes or dimensions. It should also be understood thatthe size of the concatemer relative to the reaction chamber willpreclude the presence of more than one concatemer per reaction chamber.

Solid Supports and Capture Beads

The solid support to which the template nucleic acids or primers arebound is referred to herein as the “capture solid support”. The solidsupport may be a wall of the reaction chamber (or well) including thesurface of the chemFET, or a bottom or side wall of the reaction chamberprovided such wall is capacitively coupled to the chemFET. If the solidsupport is a bead, then such bead may be referred to herein as a“capture bead”. Such beads are generally referred to herein as “loaded”with or “bearing” nucleic acid if they have nucleic acids attached totheir surface (whether covalently or non-covalently) and/or present intheir interior core. Some capture beads comprise a porous surface thatallows entry and exit of small compounds such as amplification orsequencing reagents (e.g., dNTPs, co-factors, etc.). This class of beadstypically will comprise nucleic acids internally and in this way theyfunction to localize the nucleic acids, optionally without the need toattach the nucleic acids to a solid support. In embodiments in whichcapture beads are used, preferably each reaction well comprises only asingle capture bead.

The degree of saturation of any capture (i.e., sequencing) bead withtemplate nucleic acid to be sequenced may not be 100%. In someembodiments, a saturation level of 10%-100% exists. As used herein, thedegree of saturation of a capture bead with a template refers to theproportion of sites on the bead that are conjugated to template. In someinstances this may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or it may be100%.

Microwell Arrays

Important aspects of the invention contemplate sequencing a plurality ofdifferent template nucleic acids simultaneously. This may beaccomplished using the sensor arrays described herein. In oneembodiment, the sensor arrays are overlayed (and/or integral with) anarray of microwells (or reaction chambers or wells, as those terms areused interchangeably herein), with the proviso that there be at leastone sensor per microwell. Present in a plurality of microwells is apopulation of identical copies of a template nucleic acid. There is norequirement that any two microwells carry identical template nucleicacids, although in some instances such templates may share overlappingsequence. Thus, each microwell comprises a plurality of identical copiesof a template nucleic acid, and the templates between microwells may bedifferent.

The microwells may vary in size between arrays. The size of thesemicrowells may be described in terms of a width (or diameter) to heightratio. In some embodiments, this ratio is 1:1 to 1:1.5. The bead to wellsize (e.g., the bead diameter to well width, diameter, or height) ispreferably in the range of 0.6-0.8.

The microwell size may be described in terms of cross section. The crosssection may refer to a “slice” parallel to the depth (or height) of thewell, or it may be a slice perpendicular to the depth (or height) of thewell. The microwells may be square in cross-section, but they are not solimited. The dimensions at the bottom of a microwell (i.e., in a crosssection that is perpendicular to the depth of the well) may be 1.5 μm by1.5 μm, or it may be 1.5 μm by 2 μm. Suitable diameters include but arenot limited to at or about 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μmor less. In some particular embodiments, the diameters may be at orabout 44 μm, 32 μm, 8 μm, 4 μm, or 1.5 μm. Suitable heights include butare not limited to at or about 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm,1 μm or less. In some particular embodiments, the heights may be at orabout 55 μm, 48 μm, 32 μm, 12 μm, 8 μm, 6 μm, 4 μm, 2.25 μm, 1.5 μm, orless. Various embodiments of the invention contemplate the combinationof any of these diameters with any of these heights. In still otherembodiments, the reaction well dimensions may be (diameter in μm byheight in μm) 44 by 55, 32 by 32, 32 by 48, 8 by 8, 8 by 12, 4 by 4, 4by 6, 1.5 by 1.5, or 1.5 by 2.25.

The reaction well volume may range (between arrays, and preferably notwithin a single array) based on the well dimensions. This volume may beat or about 100 picoliter (pL), 90, 80, 70, 60, 50, 40, 30, 20, 10, orfewer pL. In important embodiments, the well volume is less than 1 pL,including equal to or less than 0.5 pL, equal to or less than 0.1 pL,equal to or less than 0.05 pL, equal to or less than 0.01 pL, equal toor less than 0.005 pL, or equal to or less than 0.001 pL. The volume maybe 0.001 to 0.9 pL, 0.001 to 0.5 pL, 0.001 to 0.1 pL, 0.001 to 0.05 pL,or 0.005 to 0.05 pL. In particular embodiments, the well volume is 75pL, 34 pL, 23 pL, 0.54 pL, 0.36 pL, 0.07 pL, 0.045 pL, 0.0024 pL, or0.004 pL. In some embodiments, each reaction chamber is no greater thanabout 0.39 pL in volume and about 49 μm² surface aperture, and morepreferably has an aperture no greater than about 16 μm² and volume nogreater than about 0.064 pL.

It is to be understood therefore that the invention contemplates asequencing apparatus for sequencing unlabeled nucleic acid acids,optionally using unlabeled nucleotides, without optical detection andcomprising an array of at least 100 reaction chambers. In someembodiments, the array comprises 10³, 10⁴, 10⁵, 10⁶, 10⁷ or morereaction chambers. The pitch (or center-to-center distance betweenadjacent reaction chambers) is on the order of about 1-10 microns,including 1-9 microns, 1-8 microns, 1-7 microns, 1-6 microns, 1-5microns, 1-4 microns, 1-3 microns, or 1-2 microns.

In various aspects and embodiments of the invention, the nucleic acidloaded beads, of which there may be tens, hundreds, thousands, or more,first enter the flow cell and then individual beads enter individualwells. The beads may enter the wells passively or otherwise. Forexample, the beads may enter the wells through gravity without anyapplied external force. The beads may enter the wells through an appliedexternal force including but not limited to a magnetic force or acentrifugal force. In some embodiments, if an external force is applied,it is applied in a direction that is parallel to the well height/depthrather than transverse to the well height/depth, with the aim being to“capture” as many beads as possible. Preferably, the wells (or wellarrays) are not agitated, as for example may occur through an appliedexternal force that is perpendicular to the well height/depth. Moreover,once the wells are so loaded, they are not subjected to any other forcethat could dislodge the beads from the wells.

The Examples provide a brief description of an exemplary bead loadingprotocol in the context of magnetic beads. It is to be understood that asimilar approach could be used to load other bead types. The protocolhas been demonstrated to reduce the likelihood and incidence of trappedair in the wells of the flow chamber, uniformly distribute nucleic acidloaded beads in the totality of wells of the flow chamber, and avoid thepresence and/or accumulation of excess beads in the flow chamber.

In various instances, the invention contemplates that each well in theflow chamber contain only one nucleic acid loaded bead. This is becausethe presence of two beads per well will yield unusable sequencinginformation derived from two different template nucleic acids.

In some embodiments, the microwell array may be analyzed to determinethe degree of loading of beads into the microwells, and in someinstances to identify those microwells having beads and those lackingbeads. The ability to know which microwells lack beads provides anotherinternal control for the sequencing reaction. The presence or absence ofa bead in a well can be determined by standard microscopy or by thesensor itself. FIGS. 61J and K are images captured from an opticalmicroscope inspection of a microwell array (J) and from the sensor arrayunderlying the microwell array (K). The white spots in both images eachrepresent a bead in a well. Such microwell observation usually is onlymade once per run particularly since the beads once disposed in amicrowell are unlikely to move to another well.

It has also been found that in the absence of flow the background signal(i.e., noise) is less than or equal to about 0.25 mV, but that in thepresence of DNA-loaded capture beads that signal increases to about 1.0mV=/−0.5 mV. This increase is sufficient to allow one to determine wellswith beads.

The percentage of occupied wells in the well array may vary depending onthe methods being performed. If the method is aimed at extractingmaximum sequence data in the shortest time possible, then higheroccupancy is desirable. If speed and throughout is not as critical, thenlower occupancy may be tolerated. Therefore depending on the embodiment,suitable occupancy percentages may be at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% of the wells. As used herein, occupancy refers to thepresence of one nucleic acid loaded bead in a well and the percentageoccupancy refers to the proportion of total wells in an array that areoccupied by a single bead. Wells that are occupied by more than one beadtypically cannot be used in the analyses contemplated by the invention.

Simultaneous Sequencing Reactions

The invention therefore contemplates performing a plurality of differentsequencing reactions simultaneously. A plurality of identical sequencingreactions is occurring in each occupied well simultaneously. It is thissimultaneous and identical incorporation of dNTP within each well thatincreases the signal to noise ratio. By performing sequencing reactionsin a plurality of wells simultaneously, a plurality of different nucleicacids are simultaneously sequenced. The methods aim to maximize completeincorporation across all microwells for any given dNTP, reduce ordecrease the number of unincorporated dNTPs that remain in the wellsafter signal detection is complete, and achieve as a high a signal tonoise ratio as possible.

Before and/or while in the wells, the template nucleic acids areincubated with a sequencing primer that binds to its complementarysequence located on the 3′ end of the template nucleic acid (i.e.,either in the amplification primer sequence or in another adaptorsequence ligated to the 3′ end of the target nucleic acid) and with apolymerase for a time and under conditions that promote hybridization ofthe primer to its complementary sequence and that promote binding of thepolymerase to the template nucleic acid. The primer can be of virtuallyany sequence provided it is long enough to be unique. The hybridizationconditions are such that the primer will hybridize to only its truecomplement on the 3′ end of the template. Suitable conditions aredisclosed in Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

It will be understood that the amount of sequencing primers andpolymerases may be saturating, above saturating level, or in someinstances below saturating levels. As used herein, a saturating level ofa sequencing primer or a polymerase is a level at which every templatenucleic acid is hybridized to a sequencing primer or bound by apolymerase, respectively. Thus the saturating amount is the number ofpolymerases or primers that is equal to the number of templates on asingle bead. In some embodiments, the level is greater than this,including at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, or more thanthe level of the template nucleic acid. In other embodiments, the numberof polymerases and/or primers may be 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or up to 100% of the number of templates on a single bead in asingle well.

Suitable polymerases include but are not limited to DNA polymerase, RNApolymerase, or a subunit thereof, provided it is capable of synthesizinga new nucleic acid strand based on the template and starting from thehybridized primer. An example of a suitable polymerase subunit for somebut not all embodiments of the invention is the exo-minus (exo⁻) versionof the Klenow fragment of E. coli DNA polymerase I which lacks 3′ to 5′exonuclease activity. Other polymerases include T4 exo⁻, Therminator,and Bst polymerases. In still other embodiments that require excision ofnucleotides (e.g., in the process of a nick translation reaction),polymerases with exonuclease activity are preferred. The polymerase maybe free in solution (and may be present in wash and dNTP solutions) orit may be bound for example to the beads (or corresponding solidsupport) or to the walls of the chemFET but preferably not to the ISFETsurface itself. The polymerase may be one that is modified to compriseaccessory factors including without limitation single or double strandedDNA binding proteins.

Some embodiments of the invention require that the polymerase havesufficient processivity. As used herein, processivity is the ability ofa polymerase to remain bound to a single primer/template hybrid. As usedherein, it is measured by the number of nucleotides that a polymeraseincorporates into a nucleic acid (such as a sequencing primer) prior todissociation of the polymerase from the primer/template hybrid. In someembodiments, the polymerase has a processivity of at least 100nucleotides, although in other embodiments it has a processivity of atleast 200 nucleotides, at least 300 nucleotides, at least 400nucleotides, or at least 500 nucleotides. It will be understood by thoseof ordinary skill in the art that the higher the processivity of thepolymerase, the more nucleotides that can be incorporated prior todissociation, and therefore the longer the sequence that can beobtained. In other words, polymerases having low processivity willprovide shorter read-lengths than will polymerases having higherprocessivity. As an example, a polymerase that dissociates from thehybrid after five incorporations will only provide a sequence of 5nucleotides in length, while a polymerase that dissociates on averagefrom the hybrid after 500 incorporations will provide sequence of about500 nucleotides.

The rate at which a polymerase incorporates nucleotides will varydepending on the particular application, although generally faster ratesof incorporation are preferable. The rate of “sequencing” will depend onthe number of arrays on chip, the size of the wells, the temperature andconditions at which the reactions are run, etc.

In some embodiments of the invention, the time for a 4 nucleotide cyclemay be 50-100 seconds, 60-90 seconds, or about 70 seconds. In otherembodiments, this cycle time can be equal to or less than 70 seconds,including equal to or less than 60 seconds, equal to or less than 50seconds, equal to or less than 40 seconds, or equal to or less than 30seconds. A read length of about 400 bases may take on the order of 30minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours,4 hours, 4.5 hours, or in some instance 5 or more hours. These times aresufficient for the sequencing of megabases, and more preferablygigabases of sequence, with greater amounts of sequence being attainablethrough the use of denser arrays (i.e., arrays with greater numbers ofreaction wells and FETs) and/or the simultaneous use of multiple arrays.

Table 3 provides estimates for the rates of sequencing based on variousarray, chip and system configurations contemplated herein. It is to beunderstood that the invention contemplates even denser arrays than thoseshown in Table 3. These denser arrays can be characterized as 90 nm CMOSwith a pitch of 1.4 μm and a well size of I m which may be used with 0.7μm beads, or 65 nm CMOS with a pitch of 1 μm and a well size of 0.5 μmwhich may be used with 0.3 μm beads, or 45 μm CMOS with a pitch of 0.7μm and a well size of 0.3 μm which can be used with 0.2 μm beads.

TABLE 3 Reaction Parameters and Read Rates. chip type A B C D Epixel/CMOS 2.8 μm/0.18 μm 5.1 μm/0.35 μm 5.1 μm/0.35 μm 9 μm/3.5 μm 9μm/0.35 μm chip size 17.5 × 17.5 17.5 × 17.5 12 × 12 17.5 × 17.5 12 × 12# possible reads 27,800,000 7,220,000 2,950,000 2,320,000 1,060,000 readlength (assumption) 400 400 400 400 400 # chips/board 4 4 4 4 4 beadload efficiency 0.80 0.80 0.80 0.80 0.80 # yielded Gbp* per run 35.6 9.23.8 3.0 1.4 # times HG** (3Gbp/HG) 11.86 3.08 1.26 0.99 0.45 *Gbp isgigabases **HG is human genome

The template nucleic acid is also contacted with other reagents and/orcofactors including but not limited to buffer, detergent, reducingagents such as dithiothrietol (DTT, Cleland's reagent), single strandedbinding proteins, and the like before and/or while in the well. In oneembodiment, the polymerase comprises one or more single stranded bindingproteins (e.g., the polymerase may be one that is engineered to includeone or more single stranded binding proteins). In one embodiment, thetemplate nucleic acid is contacted with the primer and the polymeraseprior to its introduction into the flow chamber and wells thereof.

The primers may be DNA in nature or they may be modified moieties suchas PNA or LNA, or they may comprise some other modification such asthose described herein, or some combination of the foregoing. It hasbeen found according to the invention that LNA-containing primers bindefficiently to DNA templates under stringent conditions and are stillable to mediate a polymerase-mediated extension.

Some reactions may be carried out at a pH equal to or greater than 7.5,equal to or greater than 8, equal to or greater than 8.5, equal to orgreater than 9, equal to or greater than 9.5, equal to or greater than10, or equal to or greater than 11. The polymerase may be one thatincorporates nucleotides into a nucleic acid at a pH of 7-11, 7.5-10.5,8-10, 8.5-9.5, or at about 9.

In some embodiments, the enzyme has high activity in low concentrationsof dNTPs. In some embodiments, the dNTP concentration is 50 μM, 40 μM,30 μM, 20 μM, 10 μM, 5 μM, and preferably 20 μM or less.

Apyrase is an enzyme that degrades residual unincorporated nucleotidesconverting them into monophosphate and releasing inorganic phosphate inthe process. It is useful for degrading dNTPs that are not incorporatedand/or that are in excess. It is important that excess and/orunincorporated dNTP be washed away from all wells after measurements arecomplete and before introduction of the subsequent dNTP. Accordingly,addition of apyrase between the introduction of different dNTPs isuseful to remove unincorporated dNTPs that would otherwise obscure thesequencing data.

Thus, according to some aspects of the invention, a homogeneouspopulation of (or a plurality of identical) template nucleic acids isplaced into each of a plurality of wells, each well situated over andthus corresponding to at least one sensor. As discussed above,preferably the well contains at least 10, at least 100, at least 1000,at least 10⁴, at least 10⁵, at least 10⁶, or more copies of an identicaltemplate nucleic acid. Identical template nucleic acids means that thetemplates are identical in sequence. Most and preferably all thetemplate nucleic acids within a well are uniformly hybridized to aprimer. Uniform hybridization of the template nucleic acids to theprimers means that the primer hybridizes to the template at the samelocation (i.e., the sequence along the template that is complementary tothe primer) as every other template/primer hybrid in the well. Theuniform positioning of the primer on every template allows theco-ordinated synthesis of all new nucleic acid strands within a well,thereby resulting in a greater signal-to-noise ratio.

In some embodiments, nucleotides are then added in flow, or by any othersuitable method, in sequential order to the flow chamber and thus thewells. The nucleotides can be added in any order provided it is knownand for the sake of simplicity kept constant throughout a run.

In some embodiments, the method involves adding ATP to the wash bufferso that dNTPs flowing into a well displace ATP from the well. The ATPmatches the ionic strength of the dNTPs entering the wells and it alsohas a similar diffusion profile as dNTPs. In this way, influx and effluxof dNTPs during the sequencing reaction do not interfere withmeasurements at the chemFET. The concentration of ATP used is on theorder of the concentration of dNTP used.

In some embodiments, the dNTP and/or the polymerase may be pre-incubatedwith divalent cation such as but not limited to Mg²⁺ (for example in theform of MgCl₂) or Mn²⁺ (for example in the form of MnCl₂). Otherdivalent cations can also be used including but not limited to Ca²⁺,Co²⁺. This pre-incubation (and thus “pre-loading” of the dNTP and/or thepolymerase can ensure that the polymerase is exposed to a sufficientamount of divalent cation for proper and necessary functioning even ifit is present in a low ionic strength environment. Pre-incubation mayoccur for 1-60 minutes, 5-45 minutes, or 10-30 minutes, depending on theembodiment, although the invention is not limited to these time ranges.

A sequencing cycle. may therefore proceed as follows washing of the flowchamber (and wells) with wash buffer (optionally containing ATP),introduction of a first dNTP species (e.g., dATP) into the flow chamber(and wells), release and detection of PPi and then unincorporatednucleotides (if incorporation occurred) or detection of solelyunincorporated nucleotides (if incorporation did not occur) (by any ofthe mechanisms described herein), washing of the flow chamber (andwells) with wash buffer, washing of the flow chamber (and wells) withwash buffer containing apyrase (to remove as many of the unincorporatednucleotides as possible prior to the flow through of the next dNTP,washing of the flow chamber (and wells) with wash buffer, andintroduction of a second dNTP species. This process is continued untilall 4 dNTP (i.e., dATP, dCTP, dGTP and dTTP) have been flowed throughthe chamber and allowed to incorporate into the newly synthesizedstrands. This 4-nucleotide cycle may be repeated any number of timesincluding but not limited to 10, 25, 50, 100, 200 or more times. Thenumber of cycles will be governed by the length of the template beingsequenced and the need to replenish reaction reagents, in particular thedNTP stocks and wash buffers.

As part of the sequencing reaction, a dNTP will be ligated to (or“incorporated into” as used herein) the 3′ of the newly synthesizedstrand (or the 3′ end of the sequencing primer in the case of the firstincorporated dNTP) if its complementary nucleotide is present at thatsame location on the template nucleic acid. Incorporation of theintroduced dNTP (and concomitant release of PPi) therefore indicates theidentity of the corresponding nucleotide in the template nucleic acid.If no dNTP has been incorporated, no hydrogens are released and nosignal is detected at the chemFET surface. One can therefore concludethat the complementary nucleotide was not present in the template atthat location. If the introduced dNTP has been incorporated into thenewly synthesized strand, then the chemFET will detect a signal. Thesignal intensity and/or area under the curve is a function of the numberof nucleotides incorporated (for example, as may occur in a homopolymerstretch in the template. The result is that no sequence information islost through the sequencing of a homopolymer stretch (e.g., poly A, polyT, poly C, or poly G) in the template.

The sequencing reaction can be run at a range of temperatures.Typically, the reaction is run in the range of 30° C. to 70° C., 30° C.to 65° C., 30-60° C., 35-55° C., 40-50° C., or 40-45° C. It ispreferable to run the reaction at temperatures that prevent formation ofsecondary structure in the nucleic acid. However this must be balancedwith the binding of the primer (and the newly synthesized strand) to thetemplate nucleic acid and the reduced half-life of apyrase at highertemperatures. The optimum temperature for the polymerase is alsoimportant as the closer the reaction is run to that temperature, thehigher the nucleotide incorporation rate will be. Bst polymerase has aoptimum temperature of about 65° C., while T4 polymerase has an optimumtemperature of about 37° C. Thus, the optimum temperature will dependupon the polymerase being used. Some embodiments use a temperature ofabout 41° C. Other embodiments use a temperature that is higherincluding for example about 45° C., about 50° C. or about 65° C. Thesolutions, including the wash buffers and the dNTP solutions, aregenerally warmed to these temperatures in order not to alter thetemperature in the wells. The wash buffer containing apyrase however ispreferably maintained at a lower temperature in order to extend thehalf-life of the enzyme. Typically, this solution is maintained at about4-15° C., and more preferably 4-10° C.

As will be appreciated all of the foregoing methods may be automatedsuch that the various biological and/or chemical reactions are performedvia robotics. In addition, the information obtained via the signal fromthe chemFET (or chemFET array) may be provided to a personal computer, apersonal digital assistant, a cellular phone, a video game system, or atelevision so that a user can monitor the progress of the sequencingreactions remotely. This process is illustrated, for example, in FIG.71.

Diffusion Control

The nucleotide incorporation reaction can occur very rapidly. As aresult, it may be desirable in some instances to slow the reaction downor to slow the diffusion of analytes in the well in order to ensuremaximal data capture during the reaction. The diffusion of reagentsand/or byproducts can be slowed down in a number of ways including butnot limited to addition of packing beads in the wells, and/or the use ofpolymers such as polyethylene glycol in the wells (e.g., PEG attached tothe capture beads and/or to packing beads). The packing beads also tendto increase the concentration of reagents and/or byproducts at thechemFET surface, thereby increasing the potential for signal. Thepresence of packing beads generally allows a greater time to sample(e.g., by 2- or 4-fold).

Data capture rates can vary and be for example anywhere from 10-100frames per second and the choice of which rate to use will be dictatedat least in part by the well size and the presence of packing beads orother diffusion limiting techniques. Smaller well sizes generallyrequire faster data capture rates.

In some aspects of the invention that are flow-based and where the topface of the well is open and in communication with fluid over theentirety of the chip, it is important to detect the released hydrogenion prior to its diffusion out of the well. Diffusion of reactionbyproducts out of the well will lead to false negatives (because thebyproduct is not detected in that well) and potential false positives inadjacent or downstream wells (where the byproduct may be detected), andthus should be avoided. Packing beads and/or polymers such as PEG mayalso help reduce the degree of diffusion and/or cross-talk betweenwells.

In addition to the nucleic acid loaded beads, each well may alsocomprise a plurality of smaller beads, referred to herein as “packingbeads”. The packing beads may be composed of any inert material thatdoes not interact or interfere with analytes, reagents, reactionparameters, and the like, present in the wells. The packing beads may bemagnetic (including superparamagnetic) but they are not so limited. Insome embodiments the packing beads and the capture beads are made of thesame material (e.g., both are magnetic, both are polystyrene, etc.),while in other embodiments they are made of different materials (e.g.,the packing beads are polystyrene and the capture beads are magnetic).

The packing beads are generally smaller than the capture beads. Thedifference in size may vary and may be 5-fold, 10-fold, 15-fold, 20-foldor more. As an example, 0.35 μm diameter packing beads can be used with5.91 μm capture beads. Such packing beads are commercially availablefrom sources such as Bang Labs.

The placement of the packing beads relative to the capture bead mayvary. Packing beads may be positioned between the chemFET surface andthe nucleic acid loaded bead, in which case they may be introduced intothe wells before the nucleic acid loaded beads. In this way, the packingbeads prevent contact and thus interference of the chemFET surface withthe template nucleic acids bound to the capture beads. A layer ofpacking beads that is 0.1-0.5 μm in depth or height would preclude thisinteraction. The presence of packing beads between the capture bead andthe chemFET surface may also slow the diffusion of the sequencingbyproducts such as hydrogen ions, thereby facilitating data capture insome embodiments. Alternatively, the packing beads may be positioned allaround the nucleic acid loaded beads, in which case they may be added tothe wells before, during and/or after the nucleic acid loaded beads. Instill other embodiments, the majority of the packing beads may bepositioned on top of the nucleic acid loaded beads, in which case theymay be added to the wells after the nucleic acid loaded beads. If placedabove the nucleic acid loaded beads, the packing beads may act tominimize or prevent altogether dislodgement of nucleic acid loaded beadsfrom wells. In still other embodiments, the reaction wells may comprisepacking beads even if nucleic acid loaded beads are not used. It is tobe understood that in other embodiments however packing beads are notrequired as there is no need to slow the diffusion of reactionbyproducts such as hydrogen ions.

In some embodiments, diffusion may also be impacted by including in thereaction chambers viscosity increasing agents. An example of such anagent is a polymer that is not a nucleic acid (i.e., a non-nucleic acidpolymer). The polymer may be naturally or non-naturally occurring, andit may be of any nature provided it does not interfere with nucleotideincorporation and/or excision and detection thereof except for slowingthe diffusion of polymerase, released hydrogen ions, PPi, unincorporatednucleotides, and/or other reaction byproducts or reagents. An example ofa suitable polymer is polyethylene glycol (PEG). Other examples includePEO, PEA, dextrans, acrylamides, celluloses (e.g. methyl cellulose), andthe like. The polymer may be free in solution (e.g., PEG, DMSO,glycerol, and the like) or it may be immobilized (covalently ornon-covalently) to one or more sides of the reaction chamber, to thecapture bead (e.g., PEG, PEO, dextrans, and the like), and/or to anypacking beads that may be present. Non-covalent attachment may beaccomplished via a biotin-avidin interaction.

The invention further contemplates in some embodiments the use ofsoluble counterions that bind to released hydrogen ions and preventtheir exit from the well. Counterions having a pKa that is close to thepH of the reaction are preferred. Examples of counterions with diffusionrates that are slower than that of protons (at both 25° C. and 37° C.)include without limitation Cl⁻, H₂PO4⁻, HCO₃ ⁻, acetate, butyrate,histidyl, formate, lactate, and the like. In some embodiments, thecounterions are free in solution while in others they are immobilized ona solid support including without limitation reaction chamber walls. Oneof ordinary skill in the art will be able to select the most appropriatecounterion and concentration based on its pKa and the pH at which thereaction is conducted, and the mobility of the counterion. It will beunderstood that various embodiments of the invention do not require theuse of counterions.

Kits

The invention further contemplates kits comprising the various reagentsnecessary to perform a sequencing reaction and instructions of useaccording to the methods set forth herein.

One preferred kit comprises one or more containers housing wash buffer,one or more containers each containing one of the following reagents:dATP buffer, dCTP buffer, dGTP buffer or dTTP buffer, dATP, dCTP, dGTPand dTTP stocks, apyrase, SSB, polymerase, packing beads, and optionallypyrophosphatase. Importantly the kits may comprise only naturallyoccurring dNTPs. The kits may also comprise one or more wash bufferscomprising components as described in the Examples, but are not solimited. The kits may also comprise instructions for use includingdiagrams that demonstrate the methods of the invention.

The following Examples are included for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES

The Examples provide a proof of principle demonstration of thesequencing of four templates of known sequence. This artificial model isintended to show that embodiments of the apparatuses and systemsdescribed herein are able to readout nucleotide incorporation thatcorrelates to the known sequence of the templates. This is not intendedto represent typical use of the method or system in the field. Thefollowing is a brief description of these methods.

Example 1 Bead Preparation Binding of Single-Stranded Oligonucleotidesto Streptavidin-Coated Magnetic Beads

Single-stranded DNA oligonucleotide templates with a 5′ Dual Biotin tag(HPLC purified), and a 20-base universal primer were ordered from IDT(Integrated DNA Technologies, Coralville, Ind.). Templates were 60 basesin length, and were designed to include 20 bases at the 3′ end that werecomplementary to the 20-base primer (Table 4, italics). The lyophilizedand biotinylated templates and primer were re-suspended in TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8) as 40 μM stock solutions and as a 400 μMstock solution, respectively, and stored at −20° C. until use.

For each template, 60 μl of magnetic 5.91 μm (Bangs Laboratories, Inc.Fishers, Ind.) streptavidin-coated beads, stored as an aqueous, bufferedsuspension (8.57×10⁴ beads/μL), at 4° C., were prepared by washing with120 μl bead wash buffer three times and then incubating with templates1, 2, 3 and 4 (T1, T2, T3, T4: Table 4) with biotin on the 5′ end,respectively.

Due to the strong covalent binding affinity of streptavidin for biotin(Kd˜10-15), these magnetic beads are used to immobilize the templates ona solid support, as described below. The reported binding capacity ofthese beads for free biotin is 0.650 pmol/μL of bead stock solution. Fora small (<100 bases) biotinylated ssDNA template, it was conservativelycalculated that 9.1×10⁵ templates could be bound per bead. The beads areeasily concentrated using simple magnets, as with the Dynal MagneticParticle Concentrator or MPC-s (Invitrogen, Carlsbad, Calif.). The MPC-swas used in the described experiments.

An MPC-s was used to concentrate the beads for 1 minute between eachwash, buffer was then added and the beads were resuspended. Followingthe third wash the beads were resuspended in 120 μL bead wash bufferplus 1 μl of each template (40 μM). Beads were incubated for 30 minuteswith rotation (Labquake Tube Rotator, Barnstead, Dubuque, Iowa).Following the incubation, beads were then washed three times in 120 μLAnnealing Buffer (20 mM Tris-HCl, 5 mM magnesium acetate, pH 7.5), andre-suspended in 60 μL of the same buffer.

TABLE 4 Sequences for Templates 1, 2, 3, and 4T1: 5′/52Bio/GCA AGT GCC CTT AGG CTT CAG TTC AAAAGT CCT AAC TGG GCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 1)T2: 5′/52Bio/CCA TGT CCC CTT AAG CCC CCC CCA TTCCCC CCT GAA CCC CCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 2)T3: 5′/52Bio/AAG CTC AAA AAC GGT AAA AAA AAG CCAAAA AAC TGG AAA ACA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 3)T4: 5′/52Bio/TTC GAG TTT TTG CCA TTT TTT TTC GGTTTT TTG ACC TTT TCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 4)

Annealing of Sequencing Primer.

The immobilized templates, bound at the 5′ end to 5.91 μm magneticbeads, are then annealed to a 20-base primer complementary to the 3′ endof the templates (Table 4). A 1.0 μL aliquot of the 400 μM primer stocksolution, representing a 20-fold excess of primer to immobilizedtemplate, is then added and then the beads plus template are incubatedwith primer for 15 minutes at 95° C. and the temperature was then slowlylowered to room temperature. The beads were then washed 3 times in 120μL of 25 mM Tricine buffer (25 mM Tricine, 0.4 mg/ml PVP, 0.1% Tween 20,8.8 mM Magnesium Acetate; ph 7.8) as described above using the MPC-s.Beads were resuspended in 25 mM Tricine buffer.

Incubation of Hybridized Templates/Primer with DNA Polymerase.

Template and primer hybrids are incubated with polymerase essentially asdescribed by Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

Loading of Prepared Test Samples onto the ISFET Sensor Array.

The dimensions and density of the ISFET array and the microfluidicspositioned thereon may vary depending on the application. A non-limitingexample is a 512×512 array. Each grid of such an array (of which therewould be 262144) has a single ISFET. Each grid also has a well (or asthey may be interchangeably referred to herein as a “microwell”)positioned above it. The well (or microwell) may have any shapeincluding columnar, conical, square, rectangular, and the like. In oneexemplary conformation, the wells are square wells having dimensions of7×7×10 μm. The center-to-center distance between wells is referred toherein as the “pitch”. The pitch may be any distance although it ispreferably to have shorter pitches in order to accommodate as large ofan array as possible. The pitch may be less than 50 μm, less than 40 μm,less than 30 μm, less than 20 μm, or less than 10 μm. In one embodiment,the pitch is about 9 μm. The entire chamber above the array (withinwhich the wells are situated) may have a volume of equal to or less thanabout 30 μL, equal to or less than about 20 μL, equal to or less thanabout 15 μL, or equal to or less than 10 μL. These volumes thereforecorrespond to the volume of solution within the chamber as well.

Loading of Beads in an ‘Open’ System.

Beads with templates 1-4 were loaded on the chip (10 μL of eachtemplate). Briefly, an aliquot of each template was added onto the chipusing an Eppendorf pipette. A magnet was then used to pull the beadsinto the wells.

Loading of Beads in a ‘Closed’ System.

Both the capture beads the packing beads are loaded using flow.Microliter precision of bead solution volume, as well as positioning ofthe bead solution through the fluidics connections, is achieved as shownin FIGS. 62-70 using the bead loading fitting, which includes a majorreservoir (approx. 1 mL in volume), minor reservoir (approx. 10 μL involume), and a microfluidic channel for handling small volumes of beadsolution. This method also leverages the microliter precision of fluidapplication allowed by precision pipettes.

The chip comprising the ISFET array and flow cell is seated in the ZIF(zero insertion force) socket of the loading fixture, then attaching astainless steel capillary to one port of the flow cell and flexiblenylon tubing on the other port. Both materials are microfluidic-typefluid paths (e.g., on the order of <0.01′ inner diameter). The beadloading fitting, consisting of the major and minor reservoirs, itattached to the end of the capillary. A common plastic syringe is filledwith buffer solution, then connected to the free end of the nylontubing. The electrical leads protruding from the bottom of the chip areinserted into a socket on the top of a fixture unit (not shown).

The chip comprising the ISFET array and flow cell is seated in a socketsuch as a ZIF (zero insertion force) socket of the loading fixture, thena stainless steel capillary may be attached to one port of the flow celland flexible nylon tubing on the other port. Both materials aremicrofluidic-type fluid paths (e.g., on the order of <0.01′ innerdiameter). The bead loading fitting, consisting of the major and minorreservoirs, it attached to the end of the capillary. A common plasticsyringe is filled with buffer solution, then connected to the free endof the nylon tubing. The electrical leads protruding from the bottom ofthe chip are inserted into a socket on the top of a fixture unit (notshown).

It will be appreciated that there will be other ways of drawing thebeads into the wells of the flow chamber, including centrifugation orgravity. The invention is not limited in this respect.

DNA Sequencing Using the ISFET Sensor Array in an Open System.

A illustrative sequencing reaction can be performed in an ‘open’ system(i.e., the ISFET chip is placed on the platform of the ISFET apparatusand then each nucleotide (5 μL resulting in 6.5 μM each) is manuallyadded in the following order: dATP, dCTP, dGTP and dTT (100 mM stocksolutions, Pierce, Milwaukee, Wis.), by pipetting the given nucleotideinto the liquid already on the surface of the chip and collecting datafrom the chip at a rate of 2.5 MHz. This can result in data collectionover 7.5 seconds at approximately 18 frames/second. Data may thenanalyzed using LabView.

Given the sequences of the templates, it is expected that addition ofdATP will result in a 4 base extension for template 4. Addition of dCTPwill result in a 4 base extension in template 1. Addition of dGTP willcause template 1, 2 and 4 to extend as indicated in Table 5 and additionof dTTP will result in a run-off (extension of all templates asindicated).

Preferably when the method is performed in a non-automated manner (i.e.,in the absence of automated flow and reagent introduction), each wellcontains apyrase in order to degrade the unincorporated dNTPs, oralternatively apyrase is added into each well following the addition andincorporation of each dNTP (e.g., dATP) and prior to the addition ofanother dNTP (e.g., dTTP). It is to be understood that apyrase can besubstituted, in this embodiment or in any other embodiment discussedherein, with another compound (or enzyme) capable of degrading dNTPs.

TABLE 5 Set-up of experiment and order of nucleotide addition. dATP dCTPdGTP dTTP T1 0 (3: C; 1 Run-off (25) 1: A)4 T2 0 0 4 Run-off (26) T3 0 00 Run-off (30) T4 4 0 2 Run-off (24)

DNA Sequencing Using Microfluidics on Sensor Chip.

Sequencing in the flow regime is an extension of open application ofnucleotide reagents for incorporation into DNA. Rather than add thereagents into a bulk solution on the ISFET chip, the reagents are flowedin a sequential manner across the chip surface, extending a single DNAbase(s) at a time. The dNTPs are flowed sequentially, beginning withdTTP, then dATP, dCTP, and dGTP. Due to the laminar flow nature of thefluid movement over the chip, diffusion of the nucleotide into themicrowells and finally around the nucleic acid loaded bead is the mainmechanism for delivery. The flow regime also ensures that the vastmajority of nucleotide solution is washed away between applications.This involves rinsing the chip with buffer solution and apyrase solutionfollowing every nucleotide flow. The nucleotides and wash solutions arestored in chemical bottles in the system, and are flowed over the chipusing a system of fluidic tubing and automated valves. The ISFET chip isactivated for sensing chemical products of the DNA extension duringnucleotide flow.

Example 2 On-Chip Polymerase Extension Detected by pH Shift on an ISFETArray

Streptavidin-coated 2.8 micron beads carrying biotinylated synthetictemplate to which sequencing primers and T4 DNA polymerase are boundwere subjected to three sequential flows of each of the fournucleotides. The template sequence downstream of the sequencing primerwas a G(C)₁₀(A)₁₀ (SEQ ID NO:5). Each nucleotide cycle consisted offlows of dATP, dCTP, dGTP and dTTP, each interspersed with a wash flowof buffer only. Flows from the first cycle are shown in blue, flows fromthe second cycle in red, and the third cycle in yellow. As shown in FIG.72A, signal generated for both of the two dATP flows were very similar.FIG. 72B shows that the first (blue) trace of dCTP is higher than thedCTP flows from subsequent cycles, corresponding to the flow in whichthe polymerase should incorporate a single nucleotide per templatemolecule. FIG. 72C shows that the first (blue) trace of dGTP isapproximately 6 counts higher (peak-to-peak) than the dGTP flows fromsubsequent cycles, corresponding to the flow in which the polymeraseshould incorporate a string of 10 nucleotides per template molecule.FIG. 72D shows that the first (blue) trace of dTTP is also approximately6 counts higher (peak-to-peak) than the dTTP flows from subsequentcycles, corresponding to the flow in which the polymerase shouldincorporate 10 nucleotides per template molecule.

Example 3 Sequencing in a Closed System and Data Manipulation

Sequence has been obtained from a 23-mer synthetic oligonucleotide and a25-mer PCR product oligonucleotide. The oligonucleotides were attachedto beads which were then loaded into individual wells on a chip having1.55 million sensors in a 1348×1152 array having a 5.1 micron pitch(38400 sensors per mm²). About 1 million copies of the syntheticoligonucleotide were loaded per bead, and about 300000 to 600000 copiesof the PCR product were loaded per bead. A cycle of 4 nucleotidesthrough and over the array was 2 minutes long. Nucleotides were used ata concentration of 50 micromolar each. Polymerase was the only enzymeused in the process. Data were collected at 32 frames per second.

FIG. 73A depicts the raw data measured directly from an ISFET for thesynthetic oligonucleotide (SE ID NO:6). One millivolt is equivalent to68 counts. The data are sampled at each sensor on the chip (1550200sensors on a 314 chip) many times per second. The Figure is color-codedfor each nucleotide flow. With each nucleotide flow, several seconds ofimaging occur. The graph depicts the concatenation of those individualmeasurements taken during each flow. The Y axis is in raw counts, andthe X axis is in seconds. Superimposed just above the X axis are theexpected incorporations at each flow.

FIG. 73B depicts the integrated value for each nucleotide flow,normalized to the template being sequenced. The integrated value istaken from the raw trace measurements shown in FIG. 73A, and theintegral bounds have been chosen to maximize signal to noise ratio. Theresults have been normalized to the signal per base incorporation, andgraphed per nucleotide flow. The Y axis is incorporation count, and theX axis is nucleotide flow number, alternating through TACG.

FIGS. 74A and 74B represent the same type of measurements taken andshown in FIGS. 73A and 73B, with the exception that the signal beingdetected here is from a PCR product (the 25-mer oligonucleotide, SEQ IDNO:7), rather than a synthetic oligonucleotide.

Improving Pixel and Array Signal-to-Noise Ratio

The reliability of signal decoding from each ISFET and from the ISFETarray as a whole is dependent on the amplitude of the signal output byeach ISFET, and its respective signal-to-noise ratio. Some changes tothe foregoing fabrication methods, judicious materials selection, andchanges to pixel and array design can be employed to increaseconsiderably the output of the ISFETs in the array and decrease variousnoise sources. That is, these changes result in a more sensitive andmore accurate sensor array. The improvements can be implementedindividually or in various combinations, and can result in significantperformance gains to the signal-to-noise ratio (SNR).

As more fully discussed below, the improvements involve: (1)over-coating (i.e., “passivating”) the sidewalls (typically formed ofTEOS-oxide or another suitable material, as above-described) and sensorsurface at the bottom of the microwells with various metal oxide or likematerials, to improve their surface chemistry (i.e., make the sidewallsless reactive) and electrical properties; (2) thinning out the coating(deposition material) on the floating gate; (3) increasing the surfacearea for charge collection at the floating gate; (4) and modified arrayand pixels designs to reduce charge injection into the electrolyte andother noise sources.

Floating Gate Deposition Layer Material and Thickness

As illustrated in FIG. 75A, it is now appreciated that if a dielectriclayer is added over the floating gate structure of the ISFET sensorarrangement, the path from the analyte to the ISFET gate may be modeledas a series connection of three capacitances: (1) the capacitanceattributable to the above-described charge double layer at theanalyte-dielectric layer interface (labeled C_(DL)), (2) the capacitancedue to the floating gate dielectric layer (C_(FGD)), and (3) the gateoxide capacitance (C_(OX)). (Note that in the text above, the floatinggate dielectric layer is sometimes referred to as a “passivation” layer.Here, we refer more specifically to the layer as a floating gatedielectric layer in order to avoid any suggestion that the materialcomposition of the layer is necessarily related to the so-calledpassivation material(s) often used in CMOS processing (e.g., PECVDsilicon nitride) to coat and protect circuit elements.) The seriescapacitance string extends between the liquid analyte 75-1 in the wellsand the ISFET gate 75-2.

It is well known that capacitances in series form a capacitive voltagedivider. Consequently, only a fraction of the signal voltage, V_(S),generated by or in the analyte, is applied to the gate oxide as thevoltage V_(G) that drives the ISFET. If we define the gate gain asV_(G)/V_(S), one would ideally like to have unity gain—i.e., no signalloss across any of the three capacitances. Of course, unity gain is notachievable, but the actual gate gain can be optimized. The value ofC_(DL) is a function of material properties and is typically on theorder of about 10-40 μF/cm². The gate oxide capacitance is typically avery small value by comparison. Thus, by making C_(FGD) much greaterthan the series combination of C_(OX) and C_(DL) (for short,C_(FGD)>>C_(OX)), the gate gain can be made to approach unity as closelyas is practical.

To achieve the relationship C_(FGD)>>C_(OX), one can minimize C_(OX),maximize C_(FGD), or both. There is not a lot that can be done to alterthe gate oxide capacitance much when using standard CMOS foundrytechniques to fabricate the ISFETs. That is, for practical reasons onemust typically accept the gate oxide capacitance value as a “given.”Thus, emphasis may be placed on maximizing C_(FGD). Such maximizationcan be achieved by using a thin layer of high dielectric constantmaterial, or by increasing the area of the floating gate metallization.Since increasing floating gate area conflicts with a goal of having ahigh density sensor array, attention has been focused on the dielectriclayer.

Materials exist and may be used that have higher dielectric constantsthan the customary CMOS gate oxide material, silicon dioxide. So, if inthe course of fabrication such a gate oxide material has been depositedonto the floating gate metallization, one may etch away that material,essentially eliminating it, and deposit a suitable high dielectricconstant floating gate dielectric layer directly onto the floating gatemetallization. Or, one may simply deposit such a floating gatedielectric layer directly onto the floating gate metallization withouthaving to etch first. In either situation, there are then only twoseries capacitances that matter between the analyte and the ISFET gate,C_(DL) and C_(FGD). Gate gain can then be maximized by makingC_(FGD)>>C_(DL). Thus, achieving a large value for C_(FGD) is desirable,while also satisfying other requirements (e.g., reliable manufacture).

The capacitance C_(FGD) is essentially formed by a parallel platecapacitor having the floating gate dielectric layer as its dielectric.Consequently, for a given plate (i.e., floating gate metallization)area, the parameters principally available for increasing the value ofC_(FGD) are (1) the thickness of the dielectric layer and (2) theselection of the dielectric material and, hence, its dielectricconstant. The capacitance of the floating gate dielectric layer variesdirectly with its dielectric constant and inversely with its thickness.Thus, a thin, high-dielectric-constant layer would be preferred, tosatisfy the objective of obtaining maximum gate gain.

One candidate for the floating gate dielectric layer material is thepassivation material used by standard CMOS foundry processes. Thestandard (typically, PECVD nitride or, to be more precise, siliconnitride over silicon oxynitride) passivation layer is relatively thickwhen formed (e.g., about 1.3 μm), and typical passivation materials havea limited dielectric constant. A first improvement can be achieved bythinning the passivation layer after formation. This can be accomplishedby etching back the CMOS passivation layer, such as by using anover-etch step during microwell formation, to etch into and consume muchof the nitride passivation layer, leaving a thinner layer, such as alayer only about 200-600 Angstroms thick. While simple, this approach isprone to wafer-to-wafer etch variations, resulting in variability in thefinal passivation layer thickness and capacitance.

Two approaches have been at least partially evaluated for etching astandard CMOS passivation layer of silicon nitride deposited oversilicon oxynitride. We call the first approach the “partial etch”technique. It involves etching away the silicon nitride layer plusapproximately half of the silicon oxynitride layer before depositing thethin-film metal oxide sensing layer. The second approach we call the“etch-to-metal” technique. It involves etching away all of the siliconnitride and silicon oxynitride layers before depositing the thin-filmmetal oxide sensing layer. Theoretical modeling indicates that thepartial etch approach should lead to an ISFET gate gain of about 0.42.This corresponds to an increase of signal level by about 50% comparedwith a non-etched passivation layer. With an ALD Ta₂O₅ thin-film sensinglayer deposited over a “partial etch,” ISFET gains from about 0.37 toabout 0.43 have been obtained empirically, with sensor sensitivities ofabout 15.02-17.08 mV/pH.

Theoretical modeling indicates that in the “etch-to-metal” approach withthe same sensing layer, an ISFET gate gain of about 0.94 should bepossible. This would correspond to a greater than three-fold increase insignal. With an imperfect etch process that does not produce a uniformetch across the surface of the floating gate, the empirically obtainedgain has only been about 0.6, corresponding to a little more thandoubling of the signal. With improved etch chemistry/process to obtain amore uniform and flat surface at the bottom of the well, a gain close tothe model 0.94 gain should be possible.

One promising approach for improving the uniformity and flatness of theetch process is to perform two or more separate etches in series—i.e.,use a multi-step etch process. A first etch step may be performed andthe progress of that etch step may be monitored optically, at one ormultiple wavelengths. When it is detected that the first step etch hasexposed a part of the underlying metal surface, the first etch processcan be stopped and a second process or step may be begun, usingconditions that will remove the dielectric material without removing(much of) the metal.

An alternative to use of the foregoing etch processes is to simplydeposit a thinner layer of dielectric (passivation) material in thefirst place, such as the indicated 200-600 Angstroms instead of the 1.3μm of the conventional CMOS passivation process. Even better performancecan be achieved with the use of other materials and depositiontechniques to form a thin dielectric layer, preferably one of relativelyhigher dielectric constant. Among the materials believed useful for thefloating gate dielectric layer are metal oxides such as tantalum oxide,tungsten oxide, aluminum oxide, and hafnium oxide, though othermaterials of dielectric constant greater than that of the usual siliconnitride passivation material may be substituted, provided that suchmaterial is sensitive to the ion of interest. The etch-to-metal approachis preferred, with the CMOS process' passivation oxide on the floatinggate being etched completely away prior to depositing the floating gatedielectric material layer. That dielectric layer may be applied directlyon the metal extended ISFET floating gate electrode. This will helpmaximize the value of the capacitance C_(FGD).

The etch-to-metal approach is preferred, with the CMOS process'passivation oxide on the floating gate being etched completely awayprior to depositing the floating gate dielectric material layer. Thatdielectric layer may be applied directly on the metal extended ISFETfloating gate electrode. This will help maximize the value of thecapacitance C_(FGD).

Among the processes which may be used for depositing a thin layer offloating gate dielectric material are reactive or non-reactivesputtering, electron cyclotron resonance (ECR), e-beam evaporation, andatomic layer deposition (ALD), though any suitable technique may beemployed. Each of the foregoing processes has well knowncharacteristics. Importantly, however, these processes differ in theirabilities to provide conformal and uniform films, which are qualitiesthat may be important for some applications. Thus, all are usable, butthey are not necessarily equally desirable. Of the four enumeratedtechniques, ALD appears to be superior with respect to the particulardesired qualities. It is good for depositing layers whose thickness canbe controlled precisely so that wafer-to-wafer repeatability is not aproblem. Also, it is a low-temperature process that does not threatenthe aluminum interconnects that typically already will have been formedon the wafer by the time the floating gate dielectric material layer isapplied. ALD, moreover, promises to enable conformal, pinhole-free andcrack-free film coverage on the well bottom, which is required; and itis compatible with extending the deposition from the well bottoms ontothe high aspect ratio (i.e., steep) well sidewalls. Covering thesidewalls with a passivation or buffering layer will render them moreinert to the analyte.

To create such structures, a layer of microwells should be formed on topof the ISFETs wherein the microwells are open at their bottoms. If thestructure is to be formed without a floating gate dielectric layer otherthan a conventional passivation material over the floating gate, thenthe passivation material preferably should be partially etched down tothe desired thinness. This alone increases the floating gate dielectriccapacitance C_(FGD) relative to C_(DL), improving gate gain. Optionally,a thin, higher-dielectric-constant layer may be deposited or otherwiseformed over such passivation material.

The deposited layer should preferably be relatively thin—e.g., onlyabout 200-600 Angstroms thick, possibly even less. As the thin layer offloating gate dielectric material is deposited over the well bottom ontothe floating gate or its immediate coating layer, it also may be allowedto deposit conformally over the well sidewalls using, for example, theaforementioned ALD process.

The potential for improvement is considerable. As a starting point,consider one standard CMOS foundry passivation material, siliconnitride, Si₃N₄. This particular material has a sub-Nernstian response topH. Consequently, the best response we have been able to measure isabout 40 mV/pH for an ISFET sensor with a silicon nitride floating gatedeposition layer (though some improvement might be obtainable withimproved nitride deposition). This is considerably less than the idealNemstian response of 59 mV/pH at 25° C. Thus, about one-third of thesignal voltage at the interface between the analyte and the floatinggate deposition layer is lost due to use of materials with so great asub-Nernstian response. Indeed, in one example, simulations indicatedthat a three-fold improvement in gate gain is possible with changes inboth floating gate deposition material and floating gate depositionlayer thickness, for the gate geometries studied. This was thencorroborated empirically with electrical test results on a 400 Angstromaluminum oxide (Al₂O₃) floating gate deposition material.

From available literature or experimentation, one can determine that inaddition to Al₂O₃, there are other metal oxides that can be substitutedfor silicon nitride at the well bottom, to obtain a closer to Nernstianresponse. For example, Table 6 compares the pH response of ISFETs withvarious floating gate deposition oxides (specifically, SiO2, Si₃N₄,Al₂O₃ and Ta₂O₅, using published data.

TABLE 6 Characteristic SiO₂ Si₃N₄ Al₂O₃ Ta₂O₃ pH range  4-10 1-13  1-13 1-13 Sensitivity (mV/pH) 23-35 (pH > 7) 46-56  53-57 56-57 37-48 (pH <7) Sensitivity (mV/pX) Na+ 30-50 5-20 2 <1 K+ 20-30 5-25 2 <1 Responsetime (95%) (s) 1 <0.1  <0.1   <0.1 (98%) (min) Undefined 4-10 2   1Drift (mV/hr, pH 1-7) Unstable   1.0 0.1-0.2 0.1-0.2

Of the four materials compared in Table 6, SiO₂ had the lowestsensitivity to pH and no linear dependence on pH. The literatureindicated that Si₃N₄ had higher sensitivities (46-56 mV/pH) butexperiments have shown its performance to be dependent on the type ofdeposition technique and oxygen content. The best reported materialswere Al₂O₃ and Ta₂O₅, which exhibited higher sensitivity in the rangesof 53-57 and 56-57 mV/pH, respectively. One other study has indicatedthat tungsten oxide, WO₃, a material with a high dielectric constant(about 300), has a sensitivity of 50 mV/pH.

Consequently, the data indicates that using a floating gate depositionmaterial such as Ta₂O₅, Al₂O₃, HfO₃ or WO₃ will result in a largersignal in response to pH changes. In other words, if it is assumed thatthe sensitivity of Ta₂O₅ is 56 mV/pH and that the Nernstian gain isdefined as the material sensitivity divided by the ideal Nernstianresponse of 59 mV/pH at 25° C., then the Nernstian gain increases from0.67 for Si₃N₄ to about 0.95-0.96 for Ta₂O₅. Thus, with Ta₂O₅, onlyabout 4-5% of the signal voltage is lost across the floating gatedeposition layer.

The deposition of a thin film floating gate deposition layer over theside walls of the microwells provides a further benefit. By coating thewalls with a material whose pKa value is more conducive to analyte pHconditions than the TEOS oxide sidewall above mentioned, the floatinggate deposition material buffers the sidewalls so that surface reactionsthere capture fewer of the protons that otherwise would be available assignal generators once they reach the gate region.

Thus, the above-taught thin-film floating gate deposition layers providea three-fold benefit: First, they enhance sensor performance at thesensor surface by providing a more reactive interface between theanalyte and the ISFET gate (or, in other words, they are moreNernstian). Second, they serve as a replacement, thinner dielectricbetween the analyte and the metal ISFET gate (if directly applied to themetal) or between the analyte and the gate oxide (if applied over a gateoxide layer), thereby increasing the coupling capacitance and gate gain.Third, if also used to cover the microwell sidewalls, as would betypical for most deposition processes, they provide buffering by coatingthe TEOS-oxide sidewalls with a material whose pKa differs moresubstantially from the analyte pH than that of the sidewall materialitself.

There are also materials such as Iridium oxide which providesuper-Nernstian responses, which can provide a still further improvementin SNR if used as the thin film floating gate deposition layer. See,e.g., D. O. Wipf et al, “Microscopic Measurement of pH with IridiumOxide Microelectrodes,” Anal. Chem. 2000, 72, 4921-4927, and Y. J. Kimet al, “Configuration for Micro pH Sensor,” Electronics Letters, Vol.39, No. 21 (Oct. 16, 2003).

FIGS. 75B-D model the dependence of gate gain on floating gatedeposition layer thickness and material, assuming use of a conventionalgate oxide, under differing conditions. The conditions for FIG. 75B aregiven in Table 7, below:

TABLE 7 Gate oxide thickness (m) 7.70E−09, typ. ISFET gate length (m)6.00E−07 ISFET gate width (m) 1.20E−06 ISFET gate area (m2) 7.20E−13ISFET gate capacitance (F) 3.23E−15 ISFET sensor plate side length (m)6.00E−06 ISFET sensor plate area (m2) 3.60E−11 ISFET sensor platecapacitance (F) 1.84E−15

The conditions for FIG. 75C are given in Table 8, below:

TABLE 8 Gate oxide thickness (m) 7.70E−09, typ. ISFET gate length (m)5.00E−07 ISFET gate width (m) 1.20E−06 ISFET gate area (m2) 6.00E−13ISFET gate capacitance (F) 2.69E−15 ISFET sensor plate side length (m)3.50E−06 ISFET sensor plate area (m2) 1.23E−11 ISFET sensor platecapacitance (F) 6.25E−16

The conditions for FIG. 75D are given in Table 9, below:

TABLE 9 Gate oxide thickness (m) 3.80E−09 ISFET gate length (m) 4.00E−07ISFET gate width (m) 7.00E−07 ISFET gate area (m2) 2.80E−13 ISFET gatecapacitance (F) 2.54E−15 ISFET sensor plate side length (m) 1.60E−06ISFET sensor plate area (m2) 2.56E−12 ISFET sensor plate capacitance (F)9.71E−17

The deposited ALD thin film layers discussed above, like all depositedthin-films, have an intrinsic stress and stress gradient resulting frommaterial properties and/or deposition conditions. These properties canaffect the adhesion of the deposited film to the underlying substrate(the floating gate metallization and microwell sidewalls). In thefabrication examples above, various metal-oxide ceramic materials are tobe deposited onto silicon dioxide (i.e., the TEOS material of themicrowells), silicon nitride (i.e., the remaining CMOS passivationmaterial that has been etched through but which is still present on thebottom of the sidewalls) and aluminum (i.e., the metal ISFET floatinggate electrode).

Some ALD processes involve depositing materials at temperatures below400° C.; others, at temperatures above 400° C. As described above, anend-of-line forming gas anneal above 400° C. may be employed as part ofthe CMOS trapped charge neutralization process. The ALD layers depositedat temperatures less than this tend to delaminate or spall off thesilicon dioxide sidewalls. It has been found empirically that Ta₂O₅(deposited at 325° C.) spalls off the well sidewalls and Al₂O₃(deposited at 460° C.) does not.

Two methods are proposed to correct this problem, as applied tofabricating an optimum microwell passivation/floating gate dielectric(protection) material into a microwell and onto an ISFET sensor gate. Ina first method, a laminated film may be used to relieve the stress inthe as-deposited metal oxide ceramic. In a second method, a glue layeris first deposited, having superior adhesion onto which a microwellpassivation/floating gate dielectric (protection) material of optimumsurface chemistry is deposited.

As an example, the laminate layer may be an approximately 400 Angstromthick structure of alternating layers of Ta₂O₅, and Al₂O₃, (forinstance, but not limited to, each about 10-20 Angstroms thick, or ofdifferent thicknesses). It is believed to be preferable to start withAl₂O₃ (as it exhibits better adhesion to oxide) and terminate withTa₂O₅, (for its superior surface chemistry). The ALD process is ideallysuited to this as film thicknesses can be controlled down to the atomiclayer (i.e., a few Angstroms) and can be switched easily from onematerial to another simply by switching the precursor gasses introducedinto the reactor system.

The overall stress of such a laminate layer would be a combination ofthe intrinsic stresses—compressive and tensile—of the individual layers.More than two materials could be used if, say, a tertiary laminate wererequired.

The “glue layer” idea is a more straight-forward implantation. First, avery thin (e.g., 50 Angstrom) layer of good adherent material (e.g.,high temperature_(Al2O3)) may be deposited and then immediatelyfollowing that, a thicker (e.g., 400 Angstrom) layer of Ta₂O₅.

Increasing Floating Gate Surface Area

The various metal oxide materials discussed above for improving thesurface properties both of the well surface and/or of the sensor surfaceat the bottom of the well are not electrically conductive. However, onecan create an extended floating gate electrode underneath such material,extending the electrically conductive properties of the ISFET gateelectrode, by first depositing and planarize-etching a thin conformalmetal coating prior to the “passivation” layer deposition. The removalvia CMP (chemical-mechanical polishing) or other etch techniques of thethin-metal from the tops of the microwells would realize discreteelectrically isolated wells having passivated gates consisting ofsubstantially the entire interior surface area of the microwellsidewalls. This would increase the available surface area of the ISFETgate several fold. Doing so would virtually eliminate “lost protons atmicrowell walls” (i.e., those protons emanating from the sequencingreaction on the bead and otherwise hitting the non-sensing microwellwall).

The extended floating gate dielectric capacitor would be formed (by,e.g., ALD) after microwell etch. Adjustments to the microwell lateraldimensions could be necessary, depending on the thickness of thethin-metal plus passivation layers being deposited, and the bead size.

FIG. 75E depicts diagrammatically two microwells 75E1 and 75E2 beingformed with such an extended floating gate structure. As will becomeclear in a moment, the structures of FIG. 75E are in a partial state ofcompletion. With reference to FIG. 75E, one possible sequence forfabricating the microwell structure with an extended gate electrodecould be as follow: After forming the layer 75E3 of material (e.g.,TEOS) which will provide the microwell walls, on top of a CMOS waferwherein the ISFET structures have been formed, the areas that willbecome the wells are etched down to the metal layer, labeled M4,constituting the floating gate of the ISFETs. (Or, alternatively, if asstated above the microwells are formed directly in the CMOS passivationlayer, layer 75E3 can be omitted and this and other passages referringto such a layer can be understood to refer instead to the passivationlayer and formation of the microwells in the passivation layer,instead.) Next, using a process such as sputtering or ALD, a thin layerof metal (e.g., about 0.25-0.50 μm of aluminum, titanium, tantalum orother suitable material) is deposited to form a layer 74E4 in contactwith the M4 layer, running over the bottom of the well and up thesidewalls. The thin metal layer will also cover the tops of the (e.g.,TEOS) material between the microwells, forming an electrical shortcircuit between the wells, which will have to be removed. Next, one mayoptionally fill the wells with a suitable material such as variousorganic fill materials that are readily available, to prevent the nextstep from leaving unwanted debris in the microwells. The next step is toemploy CMP or another suitable technique to planarize the top of thestructure down to a level that exposes the tops of the microwells andthe material layer 75E4 on the top of the sidewalls. Having thus exposedthe metal layer 75E4, that metal is removed from the tops of themicrowell array (especially the tops of the sidewalls and TEOS betweenthe sidewalls) in any satisfactory way). For example, the metal 75E4′ atthe top of the microwell structure (i.e., on the exterior top of thesidewalls) may be etched away or removed using metal CMP to furtherplanarize the tops of the wells. Having removed the short circuitbetween the wells, the filler material, if used, is then removed. Theextended gate metallization is then covered by application of the thindielectric layer discussed above, 75E5, such as a tantalum pentoxide(Ta₂O₅) ALD passivation, using ALD, for example. Thedielectric/passivation must cover all edges of layer 75E5 to avoidelectrically short-circuiting wells via the analyte fluid.

As an alternate fabrication process, the removal of material 75E4′ couldbe done by patterning an inverse of the microwell pattern as a mask(i.e., opening the areas between the wells) and then using a standardmetal etch.

The collection of all charge that reaches the microwell sidewalls, aswell as its bottom, renders the pixels more sensitive to the reaction inthe wells.

Another way to improve charge collection and sensitivity is to employfor the surfaces contacting the electrolyte a material that has a pointof zero charge that matches the operating pH of the analyte.

Improved on-Chip Electronics

There are two key areas where the on-chip electronics can potentially beimproved to increase the voltage signal gain and to reduce noise: thepixel circuit and the readout circuit.

Pixel Circuit

In a basic pixel circuit such as is illustrated above in FIG. 9, forexample, the bulk potential of the ISFET 150 is taken to the highestcircuit potential. Unfortunately, the threshold voltage VT of the deviceis affected by the potential difference between the source and bulk,VSB. This phenomenon is known as the body effect and is modeled as:

V _(T) =V _(T0)+γ(√(2|φ_(F) |+V _(SB))−√2|φ_(F)|

where VT0 is the threshold voltage when the source voltage is equal tothe bulk potential, 2 is the surface potential at threshold, and γ isthe body effect coefficient. Consequently, the threshold voltage willvary due to the body effect; and the ISFET source gain, defined as theratio VS/VG, will be less than the ideal value of unity. Although itcannot be measured directly, it is thought that the ISFET source gain isin the neighborhood of 0.9. In other words, up to 10% of the maximumvoltage signal that could be measured may be lost due to the bodyeffect.

If each ISFET is placed in its own n-well, and the source and bulkterminals are connected-together, then the body effect can be eliminatedand an ISFET source gain of unity can be realized. Furthermore, if eachISFET is isolated from the rest of the chip by a reverse-biased diodebetween the n-well and the substrate, then the device will be lesssusceptible to substrate noise. In other words, the total ISFET noiseshould be lower if the device is located in its own n-well.

Reducing Injection of Noise into Electrolyte

A second aspect to improving the SNR is that of reducing noise. A majorcomponent of such noise is noise that is coupled into the analyte fluidby the pixels in every column of the array, due to the circuit dynamics.Two noise injection mechanisms have been identified: the drain sidecolumn buffer injects noise through each pixel and each row selectionpumps charge into the fluid. These mechanisms are focused on the ISFETdrain and on the ISFET source.

The ISFET Drain Problem

When a row is selected in the array, the drain terminal voltage sharedbetween all of the ISFETs in a column moves up or down (as a necessaryrequirement of the source-and-drain follower). This changes thegate-to-drain capacitances of all of the unselected ISFETs in thecolumn. In turn, this change in capacitance couples from the gate ofevery unselected ISFET into the fluid, ultimately manifesting itself asnoise in the fluid (i.e., an incorrect charge, one not due to thechemical reaction being monitored). That is, any change in the shareddrain terminal voltage can be regarded as injecting noise into the fluidby each and every unselected ISFET in the column. Hence, if the shareddrain terminal voltage of the unselected ISFETs can be kept constantwhen selecting a row in the array, this mechanism of coupling noise intothe fluid can be reduced or even effectively eliminated.

The ISFET Source Problem

When a row is selected in the array, the source terminal voltage of allof the unselected ISFETs in the column also changes. In turn, thatchanges the gate-to-source capacitance of all of these ISFETs in thecolumn. This change in capacitance couples from the gate of everyunselected ISFET into the fluid, again ultimately manifesting itself asnoise in the fluid. That is, any change in the source terminal voltageof an unselected ISFET in the column can be regarded as an injection ofnoise into the fluid. Hence, if the source terminal voltage of theunselected ISFETs can be kept when selecting a row in the array, thismechanism of coupling noise into the fluid via can be reduced or eveneffectively eliminated.

A column buffer may be used with some passive pixel designs to alleviatethe ISFET drain problem but not the ISFET source problem. Thus, a columnbuffer most likely is preferable to the above-illustratedsource-and-drain follower. With the illustrated three-transistor passivepixels employing a source-and-drain follower arrangement, there areessentially two sense nodes, the ISFET source and drain terminals. Byconnecting the pixel to a column buffer and grounding the drain terminalof the ISFET, there will be only one sense node: the ISFET sourceterminal. So the drain problem is eliminated.

Active Pixel Design

All of the above-discussed passive pixels circuits present noise andscalability challenges. That is, increasing the size of the arraytypically leads to increased bus capacitance and a non-linear increasein power needs. Increasing readout speed comes at the expense ofincreased readout noise. Replacing passive ISFET pixels with activepixels, each having an active amplifier transistor as an integralelement, can reduce noise coupled into the fluid, along with reducingreadout noise, low frequency noise and fixed pattern noise. Thisapproach, moreover, appears to provide a low-noise ISFET pixel thatsuccessfully eliminates both the ISFET drain problem and the ISFETsource problem, the latter because the sense node (i.e., ISFET sourceterminal) is decoupled from the column bus.

Note that, to make a measurement, the sense node has to be connected toa current source, with current flowing. However, switching the currentsource off and on introduces a disturbance at its own to the sense node,and thus couples noise into the fluid. To avoid this problem and furtherimprove the signal-to-noise ratio, a single transistor current sourcecan be introduced into each active ISFET pixel. Current then would beflowing through every pixel in every column of the array all of thetime. Of course, there are obvious implications for power consumptionand it would be advisable to operate this current source transistor insub-threshold mode to minimize power consumption.

Turning to FIG. 75F, there is shown a first example 75F1 of such anactive pixel. The active pixel 75F1 has four transistors, 75F2-75F5, ofwhich 75F2 is the ISFET, its floating gate being shown diagrammaticallyat 75F6. While the column bus 75F7 is decoupled from the sense node bysource follower transistors 75F4 and 75F5 to reduce readout noise,switching the current source (not shown) on and off with transistor 75F3introduces a disturbance at the sense node (the source of ISFET 75F2)which couples noise into the fluid.

A second example of a four-transistor active pixel 75G1 is shown in FIG.75G. This pixel uses a single-MOSFET current source 75G3 to avoidintroducing a disturbance at the sense node. The current sourcetransistor can be operated as a reverse-biased diode (cutoff) or insub-threshold mode to minimize power consumption.

By sharing some transistors between pixels, the average number oftransistors per pixel, and hence pixel size, can be reduced. Forexample, see the arrangement of FIG. 75H, wherein transistors 75H1 and75H2 are shared between four pixels 75H3-74H6, resulting in an averageof 3.5 transistors per pixel.

An example of a six-transistor active pixel 75I0 is shown in FIG. 75I.In operation, the reset input 75I1 is enabled, transistor 75I3 is turnedon and the resultant voltage from ISFET 75I2 is measured by the sourcefollower transistors 75I4-75I5. The difference between the reset leveland the signal level from ISFET 75I2 is the output of the sensor.

By taking two samples per pixel (i.e., using correlated double sampling(CDS), the six transistor pixel 75I0 can suppress 1/f noise, and fixedpattern noise due to threshold voltage variations.

As shown in FIG. 75J, the concept of sharing transistors among a groupof pixels also can be applied to the six-transistor pixel example ofFIG. 75I. By sharing three transistors 75I1, 75I4 and 75I5, four pixels75J1-75J4 can have an average of 3.75 transistors each. The two sharingexamples use four pixels but there is no reason transistors cannot beshared among a different number of pixels.

Moving from a passive pixel design to an active amplified pixel designthus improves the scalability of the design and reduces the readoutnoise. A single-MOSFET current source is required in each ISFET pixel toavoid coupling noise into the fluid. By increasing the number oftransistors per pixel, correlated double sampling can be used at thepixel level to reduce flicker noise and fixed pattern noise. Further,the “shared pixel” concept can be used to reduce the effective number oftransistors per pixel to achieve a smaller pixel size.

To reduce power consumption, the FETs (or selected ones of them) can beoperated in the so-called “weak inversion” or “sub-threshold” mode.

Readout Circuit

The above-described readout circuit, which comprises bothsample-and-hold and multiplexer blocks, also has a gain that is lessthan the ideal value of unity. Furthermore, the sample-and-hold blockcontributes a significant percentage of the overall chip noise, perhapsmore than 25%. From switched-capacitor theory, the sample-and-hold“kT/C” noise is inversely proportional to capacitance. Hence, bychoosing a larger capacitor, the sample-and-hold noise can be reduced.Another approach to reducing noise is to employ Correlated DoubleSampling (CDS), where a second sample-and-hold and difference circuit isused to cancel out correlated noise. This approach is discussed atgreater length, below.

Correlated Double Sampling

Correlated Double Sampling (CDS) is a known technique for measuringelectrical values such as voltages or currents that allows for removalof an undesired offset. The output of the sensor is measured twice: oncein a known condition and once in an unknown condition. The valuemeasured from the known condition is then subtracted from the unknowncondition to generate a value with a known relation to the physicalquantity being measured. The challenge here is how to be efficient inimplementing CDS and how to address both correlated noise and theminimization of noise injection into the analyte fluid.

A starting point is the sensor pixel and its readout configuration asexpressed in earlier parts of this application. Referring to FIG. 77A,the basic passive sensor pixel 77A1 is a three-transistor arrangement ofan ISFET 77A2 and a pair of row select transistors, 77A3 and 77A4connected to the ISFET source. Transistor 77A3 is connected in turn to acurrent source or sink 77A5. A readout is obtained via transistor 77A4which is connected to the input of sense amplifier 77A6. Adiode-connected transistor 77A7 in series with another amplifier, 77A8,connects in a feedback loop from the output of the sense amplifier tothe drain of the ISFET. The sense amplifier output is captured by asample-and-hold circuit 77A9, which feeds an output amplifier 77A 10.

As discussed above, the voltage changes on the ISFET source and draininject noise into the analyte, causing errors in the sensed values. Twoconstructive modifications can reduce the noise level appreciably, asshown in FIG. 77B.

The first change is to alter the signals on the ISFET. The feedback loopto the drain of the ISFET is eliminated and the drain is connected to astable voltage, such as ground. A column buffer 77B is connected to theemitter of transistor.

The second change is to include a circuit to perform CDS on the outputof the column buffer. As mentioned above, CDS requires a first,reference value. This is obtained by connecting the input of columnbuffer 77B1 to a reference voltage via switch 77B2, during a first, orreference phase of a clock, indicated as the “SH” phase. A combined CDSand sample-and-hold circuit then double samples the output of the columnbuffer, obtaining a reference sample and a sensed value, performs asubtraction, and supplies a resulting noise-reduced output value, sincethe same correlated noise appears in the reference sample and in thesensor output.

The operation of the CDS and sample-and-hold circuit is straightforward.The circuit operates on a two-phase clock, the first phase being the SHphase and the second phase being the SHb phase. Typically, the phaseswill be symmetrical and thus inverted values of each other. Thereference sample is obtained in the SH phase and places a charge (andthus a voltage) on capacitor Cin, which is subtracted from the output ofthe column buffer when the clock phase changes.

An alternative embodiment, still with a passive sensor pixel, is shownin FIG. 77C. The sensor pixel in this embodiment is a two-transistorcircuit comprising ISFET whose drain is connected to a fixed supplyvoltage, VSSA. There is no transistor comparable to 77A4, and the pixeloutput is taken from the emitter of transistor 77A3, instead. The CDSand sample-and-hold circuit has been simplified slightly, by theelimination of a feedback loop, but it serves the same function, inconjunction with the charge (voltage) stored on capacitor Cbl, ofsubtracting a reference value on capacitor Cin from the signal suppliedby the sensor pixel.

Digital Pixels and Readouts

As signal-to-noise ratios often can be improved by moving from theanalog domain into the digital domain, we have also begun to explore thepossibilities for creating digital ISFRT pixels and digital pixelreadouts.

Consider first the architecture shown in FIG. 75K. There, a singleanalog-to-digital converter (ADC) 75K1 converts the analog output of acolumn addressing circuit 75K2, supplying output from pixel array 75K3,to digital form. Low fixed pattern noise is achievable but the operationof this architecture is low, the single ADC being a bottleneck. Theframe rate is limited by the number of pixels and the time required forthe ADC to complete one conversion. Thus, this architecture is notsuitable for high resolutions.

To achieve higher throughput (i.e., frame rate), one ADC 75L11-75L1 nmay be used for each column of the array 75K3, as illustrated in FIG.75L. Indeed, the frame rate can be nearly n times faster. Instead of theADC being a speed-limiting factor, frame rate can be limited by theoutput transfer capabilities of the array. The down-side, of course, isthat power consumption is increased.

In either of these two cases, parallelism and frame rate can beincreased by dividing the array into two groups (75M1, 75M2 in FIGS. 75Mand 75N1, 75N2 in FIG. 75N), and reading out each group separately.Again, however, there is a power consumption penalty to be paid. Notillustrated in FIGS. 75M and 75N is a multiplexer which may be used, ifdesired to provide a single output stream (e.g., interleaving outputs ofthe first, or top, group with outputs of the second, or bottom, group).

To go more directly into the digital domain, one has to move fromconverting an analog array output into generating a digital outputdirectly at each pixel. In general, this requires providing at eachpixel some form of analog-to-digital conversion, and memory (at least1-bit, for each). Converting the analog sensor signal to digital on an“in-pixel” basis creates an opportunity to achieve the largest possiblesignal-to-noise ration (SNR). It also is inherently scalable, allowinghigh speed, massively parallel readout of digital sensor data, with theframe rate limitations being dominated by array input/output (I/O)transfer speed, owing to the fact that all pixels are converting sensedvalues to digital form in parallel.

A basic digital pixel architecture 75O1 is as shown in FIG. 75O. Itincludes an ISFET 75O2, a current source 75O3, an ADC 75O4 and memory75O5, whose operation is as discussed above. As with the above-discussedactive ISFET pixels, the circuitry around the sense node (not shown indetail here, for clarity) preferably is chosen to avoid coupling noiseinto the fluid.

The concept of sharing circuitry between multiple pixels, to reduce theaverage chip area per pixel and to reduce the average and total powerconsumption, can be extended to digital pixel architectures, as well.For example, FIG. 75P depicts an ADC 75P1 and memory 75P2 being sharedby four ISFET cells 75P3-75P6 or pixels (here using that term eventhough the ADC and memory are shared and not part of the cells withinthe dotted lines).

With such digital pixels formed into an array, from a readoutperspective the array resembles a memory array. Thus, as shown in FIG.75Q, when the individual pixels provide digital output values, rowaddressing circuitry 75Q1 and column sense amplifiers 75Q2 can providethe readout functionality from pixel array 75Q3, as they would in amemory array.

The approaches of FIGS. 75M and 75N—i.e., subdividing or segmenting thearray and processing separately the subdivisions/segments—may beimplemented with any suitable pixel architecture, such as those of FIGS.77B and 77C, with digital or analog pixel output. For example, theoutput of the column mux 77B4 or 77C4 of each segment can be an input toa further multiplexer, not shown for selecting between segments tosupply a common output.

Thus, a row and column addressing scheme allows selection of a variablysized sub-region within the array. This facilitates a trade-off of thesize of the array being interrogated with the readout speed (i.e. framesper second). A faster sample rate can have a number of potentialadvantages:

1). A faster sample rate when combined with a digital filter can producebetter signal-to-noise measurements for the pixels within thesub-region. For example, selecting a sub-region of one-fourth the arraysize would allow sample rates approximately four times higher for thepixels within the sub-region. A simple filter that averages fourconsecutive samples together would reduce the final sample rate backdown to the nominal whole-array frame rate, but each measurement wouldonly have approximately half the noise content.

2). The faster sample rate can be used to examine higher-frequencysignals than would otherwise be possible at the nominal whole-arrayframe rate of the device. For example, selecting a sub-region ofone-fourth the array size would allow sample rates approximately fourtimes higher and the bandwidth limit for measured signals would beincreased by a factor of four.

3). Both cases can be combined to provide both high-frequency responseand higher SNR. For example, selecting a sub-region one-sixteenth thewhole-array size would allow for both a two-fold increase in SNR and afour-fold increase in bandwidth.

In some applications, sensitivity and/or signal bandwidth may be moreimportant than the number of active pixels. The availability of variableframe size (which might also be called flexible bandwidth allocation, orperhaps dynamic bandwidth allocation) is valuable in these situations.

With an appropriately segmented array, it would also be possible toperform a ‘rolling’ sequencing reaction across a large array. One would*slowly* flow dNTP across a large chip. As the wave of dNTP flows acrossthe chip slowly, the sequencing reaction would only be occurring in asmall region along the ‘front’ of the dNTP flow. In theory, it would bepossible to synchronize sub-region oversampling with the dNTP front toget very accurate measurements of the entire array

Protection Diodes

To reduce possible gate oxide degradation during plasma processing(e.g., plasma etch, sputtering, PECVD, etc.), a well diode and/or asubstrate diode may be employed, as illustrated in FIGS. 75R-75T.

In FIG. 75R, diode 75R1 between the gate 75R2 of ISFET 75R3 and the well75R4 limits the voltage that can build up on the gate relative to thewell. The “overhead” added in the form of real estate occupied is quitesmall. A typical ISFET, for example, might be 1.2×0.5 μm, and the diodemight have a perimeter of about 2.8 μm and occupy only 0.49 μm².

In FIG. 75S, diode 75S1 between the gate 75R1 of ISFET 75R3 and thesubstrate 75S2 limits the voltage that can build up on the gate relativeto the substrate. The “overhead” added in the form of real estateoccupied is quite small. A typical ISFET, for example, might be 1.2×0.5μm, and the diode might have a perimeter of about 2.8 μm and occupy only0.49 μm².

As shown in FIG. 75T, both diodes can be used together and their totalarea might typically be only about 0.98 μm².

Reference Electrode Alternatives: The Fluid-Fluid Interface

Performance of the instrument, over all, also can be enhanced withfurther attention to the reference electrode. With the above-described“simple” electrodes involving a metal tube, wire, etc. inserted into theflow cell, it has been observed that the reference potential introducedinto the flow cell by such electrodes is not stable. It is sensitive tovariations in fluid composition and pH. Accordingly, attention also hasbeen given to devising an improved electrode arrangement, which canintroduce a more stable reference potential.

With the simple electrode designs discussed above, the fluid-electrodeinterface influences the way the reference potential is transmitted intothe fluid. That is, the interface potential between the fluid and theelectrode fluctuates with the composition of the fluid (which may besomewhat turbulent and inhomogeneous), introducing a voltage offset tothe potential of the bulk fluid which varies with time and possiblylocation, as well.

Considerably greater reference potential stability may be achieved bymoving the location of the reference electrode so that it issubstantially isolated from changes in fluid composition. This may beaccomplished by introducing a conductive solution of a consistentcomposition over at least part of the surface of the electrode(hereafter the “electrode solution”), arranging the electrode to avoidit coming into direct contact with the fluid in the flow cell and,instead, arranging the electrode solution (not the electrode) to comeinto electrical contact with the fluid in the flow cell. The result is atransfer of the reference potential to the flow cell solution (be it areagent or wash or other solution) that is considerably more stable thanis obtained by direct insertion of an electrode into the flow cellsolution. We refer to this arrangement as a liquid-liquid or fluid-fluidreference electrode interface.

The fluid-fluid interface may be created downstream from the flow cell,upstream from the flow cell, or in the flow cell. Examples of suchalternative embodiments are shown in FIGS. 76A-76D.

Turning first to FIG. 76A, there is shown a diagrammatic illustration ofan embodiment in which the fluid-fluid interface is created downstreamfrom the flow cell. In this example, the flow cell apparatus 76A1 is, asabove, mounted on a chip 76A2 which contains the sensor array (notshown). The flow cell apparatus includes an inlet port 76A3 and anoutlet port 76A4. That is, the reagent fluids are introduced into port76A3 via conduit 76A4 and they exit via port 76A4. A first port 76A6 ofa fluid “Tee” connector 76A7 is coupled onto flow cell outlet port 76A4via conventional couplings to receive the fluid exiting from the flowcell. A reference electrode such as a hollow electrically conductivetube 76A8 is fed into another port of the Tee connector via afluid-tight coupling 79A9. The reference electrode is connected to areference potential source 76A10 and a suitable electrode solution 76A11is flowed into the center bore of the electrode tube.

Two modes of operation are possible. According to a first mode, theelectrode solution may be flowed at a rate that is high enough to avoidbackflow or diffusion from the fluid flowing out of the flow cell.According to a second mode, once the electrode solution has filled theelectrode and come into contact with the outlet flow from the flow cell,a valve (not shown) may be closed to block further flow of the electrodesolution into the electrode and, as the electrode solution is anincompressible liquid, there will be substantially no flow into or outof the electrode, yet the fluid-fluid interface will remain intact. Thispresumes, of course an absence of bubbles and other compressiblecomponents. For a fluid-fluid interface to take the place of ametal-fluid interface, the tip 76A 12 of the electrode 76A8 ispositioned to stop within the Tee connector short of the fluid flow outof the flow cell, so that it is the “electrode solution,” not theelectrode itself, that meets the outlet flow from the flow cell,indicated at 76A 13, and carries the reference potential from theelectrode to the reagent solution exiting the flow cell. The two fluidstreams interact in the Tee connector at 76A13 and if the electrodesolution is flowing, it flows out the third port 76A14 of the Teeconnector with the reagent flow, as a waste fluid flow, for disposal.

This approach eliminates interfacial potential changes at the electrodesurface.

Using a fluid-fluid interface to convey a stable reference potentialfrom a reference electrode to a flow cell, various alternativeembodiments are possible.

In one alternative, illustrated in FIG. 76B, the referencing junction(i.e., the fluid-fluid interface) can be moved into the structure of themembers forming the flow cell or even into the sensor chip itself, butwith the electrode solution never entering the flow cell. For example, amanifold 76B1 may be formed in the flow cell assembly outside the flowchamber itself, having an inlet 76B2 for receiving electrode solutionand an outlet 76B3 in fluid communication with the flow cell's outletconduit 76A4. The electrode may be a separate element disposed in themanifold or it may be a metallization applied to an interior surface ofthe manifold.

Alternatively, the manifold can be formed in the substrate of the chipitself by fabricating in the substrate a hollow region which can serveas a conduit allowing fluid passage from an inlet end to an outlet end.An electrode may be inserted therein via a separate inlet port 76B2 orpart of the (interior or exterior, as appropriate) surface of theconduit may be metalized during fabrication, to serve as the electrode.The flow path for reagent fluid to exit the flow chamber may include aconduit portion and the electrode conduit/manifold may deliver electrodesolution to the reagent fluid outlet conduit, wherein the two fluidscome into contact to provide the fluid-fluid interface that applies thereference electrode voltage to the flow cell.

In each instance, the electrode may be hollow and have the electrodesolution delivered through its interior, or the electrode solution maybe delivered over the exterior of the electrode. For example, as shownin FIG. 76B, the electrode may be hollow, such as being the interiorsurface of the manifold 76B1, and it may have an exterior that isinsulated from the flow cell using any suitable structure and material(not shown, to avoid obfuscation of the basic idea).

The electrode assembly thus may be built into the sensor chip itself orinto the flow cell or its housing, coupled with a fluid inlet throughwhich electrode solution may be introduced. The flow path for reagentfluid to exit the flow chamber may include a conduit portion 76A4 intowhich the electrode solution is presented, and wherein the two fluidflows come into contact to provide the fluid-fluid interface. Theelectrode solution may flow or be static.

As a further alternative embodiment, depicted in FIG. 76C, the electrodestructure may be integrated into or disposed within the flow cellitself. This may be done in two distinctly different ways. First, theelectrode solution may be introduced into the flow chamber and flowedfrom an inlet 76B4 into the flow cell (provided for that purpose) to anoutlet port 76A4 through which both the electrode solution and thereagent flow exit the flow chamber. If both fluids are arranged to movethrough the chamber in a laminar flow, they will not intermix (or therewill be little mixing and interaction) until they reach the outlet. Sothere need not be a barrier between the two fluids. Their entire regionof contact will be the locus of fluid-fluid interfacing, which mayprovide considerably more surface for that interface than the otherillustrated alternatives. Second, a fluid conduit may be providedadjacent to the flow chamber or even fully or partly within the flowchamber, with a non-conductive exterior. The electrode may extend alongthe interior of the conduit, between an electrode fluid inlet and afluid outlet that permits the electrode solution to interface with thereagent flow, such as in a common outlet conduit 76A4.

In the foregoing examples, the reference potential is introduced eitherin or downstream of the flow cell. However, the same approach ispossible with the electrode provided upstream of the flow cell, as showndiagrammatically in FIG. 76D. There, 76A3 is the inlet port to the flowcell and 76A4 is the outlet port, as in FIG. 76A. A cross-connector 76D1having four ports has a first port 76D2 coupled onto the inlet port. Asecond port, 76D3, receives the solution to be reacted or measured(e.g., a reagent) via inlet conduit 76A5. A third port, 76D4, is used asa waste outlet port. The fourth port, 76D5, receives the electrode inthe same manner as previously shown in FIG. 76A. Within thecross-connector, the electrode solution and the solution to bereacted/measured interact to transmit the reference potential into theflow cell. In contrast with some of the other alternative embodiments,however, at least some implementations of this embodiment may requirethat the solution to be measured/reacted must have a sufficiently highflow rate as to prevent flow of the electrode solution into the flowchamber. However, with judicious configuring of the cross-connector, itmay still be possible to avoid the need to flow electrode solutioncontinuously.

Further Developments in Fluidics

The delivery of multiple reagent solutions (and wash solutions) insequence to a common volume (i.e., flow cell or flow chamber) requiresselective switching (i.e., multiplexing) the fluid flows. Themultiplexing of fluid flows typically introduces characteristics thatare undesirable in that they produce less than ideal results, includingpotential contamination of reagents, for example, and intervals duringwhich sensor response is unusable or unreliable, reducing potentialthroughput. The volume of interest, specifically where various reagentsmust commonly flow to reach the flow cell, is relatively large. Thiscompetes with the requirement of cleanliness, as a previously flowingreagent must be completely washed out of the common volume before thenext reagent can flow through it to the flow cell. This takes time andconsumes wash solution. The characteristic of high volume usually stemsfrom the bulk of valve mechanics that is used to operate themultiplexing action. The presence of valve mechanisms in or near thecommon volume also competes with the requirement of cleanlinessdirectly, as the valves often present high surface area and/orcrevice-type volumes that can retain unwanted reagents. Hence, it wouldbe desirable to provide an improved switching mechanism for reagentflow, to reduce the time required for switching fluids and to minimizecross-contamination.

As exemplified in the embodiment illustrated in FIGS. 78A-78E, insteadof multiplexing multiple reagents right at the location of valves usedto control their flow, the reagents may be multiplexed downstream of thevalving, within a passive micro-fluidic multiplexer circuit that acts asa kind of union. The challenge of presenting a union to multiplereagents is to deliver only a single selected reagent to the chip (i.e.,flow cell) input, while having no diffusion-transported effluent fromany other reagent input. A simple nodal junction would not satisfy thisrequirement, as all incoming reagent lines, not being shuttered byvalves directly at the junction, could freely diffuse into one another.The disclosed fluidic circuits overcome this difficulty by employinglaminar flow or fluid resistance networks to discard diffuse effluent toa waste location.

The multiplexer circuit comprises a (optional) housing 778A 1 supportinga fluid multiplexer member 78A2 and having reagent input ports78A3-78A6, a wash input port 78A7, a waste output port 78A8, a chip(flow cell) output port 78A9, a wash solution inlet port 78A10, amulti-use central port 78A11 and a multi-purpose outlet supply port778A12. (Each reagent is treated in like fashion and the structure ofthe multiplexer member is the same for each reagent and for the washsolution, so the pertinent structure will be discussed in detail onlyfor one reagent, it being understood that such discussion applies aswell to the structures for the other solutions.) Each reagent inputfeeds into the underside of a corresponding curved (e.g., semi-circular)laminar channel such as channel 78B3, in FIG. 78B. Channel 78B3 may, forexample, be on the order of 0.5 mm on a cross-sectional side and acurvature 5 mm in diameter. The ends of the laminar channel feed intotwo restriction channels (e.g., 78B3-a and 78B3-b), with reduced crosssectional area and length of approximately 1 cm. A first one of therestriction channels, 78B3-a, connects to an outer, circular channel,78B10, which feeds the fluid flow to the waste outlet 78A8. The secondof the restriction channels, 78B3-b, leads to directly to a port 78A11in the center of the structure, extending downwardly in the drawing.Referring to FIGS. 78C and 78D (which show the multiplexer in reflectionrelative to FIGS. 78A and 78B), port 78A11 connects to a first leg 78B14of a T-shaped conduit structure 78B16, which has a second leg 78B18 thatreceives the wash solution input and a third leg 78B20 that suppliessolutions to the flow cell fluid input.

On each of the solution feeds, a two-way valve is employed (not shown),upstream of the multiplexer member.

There are two modes of operation for the multiplexer circuit. In a firstmode, a reagent is introduced via the multiplexer to the chip. In asecond mode, a wash solution clears the multiplexer and the chip.

In the first mode, the upstream valve on the wash solution input isturned off and no wash solution flows into conduit leg 78B18. Selectionof a particular reagent is performed by opening its associated upstreamvalve. Downstream valves (also not shown, to avoid obfuscation) for boththe chip and waste outlets are also opened. Two basic processescommence: a) referring to FIGS. 78C and 78E, the selected reagententering via 78B3 is driven to the waste output in the multiplexercircuit, traveling in opposing directions from its point of entry andthrough both restriction channels 78B3-a and 78B3-b, and b) sometypically smaller percentage of the reagent flows downward into port78A11 into conduit 78B14 and thence upward into port 78A12 and throughthe chip output port 78A9 toward the chip. The restriction channels areintended to dominate the resistance of the system, albeit quite small,such that the reagent flow to waste is balanced between the two paths.While reagent traverses the laminar channel, it is possible for effluentfrom the other reagent inputs to diffuse into the reagent stream.However, as illustrated in FIG. 79, this diffuse effluent 79-2 remainsin the lower lamina of the stream and continues on to the waste outputalong with the flow occurring via channel 78B3-b. That is, with no flowsentering the other fluid inlet ports, the solution entering port 78A3,into channel 78B3, flows from channel 78B3-b to the waste port via thesemicircular channels associated with each of the other inlet ports,sweeping out solutions that otherwise might diffuse into the incomingfluid. At the same time, a portion of the incoming reagent is directedtoward the chip from the upper lamina, 79-4, where the concentration ofeffluent is close to zero, via aperture 78B25 into port 78A11 and thenceas above described. A side view of this phenomenon is illustrated inFIG. 79.

Between reagent flows, wash solution is fed into the circuit, in thesecond mode. FIG. 78D illustrates the wash flow. Wash solution flowsboth to the chip via ports 78A10 and 78A9, and through the laminarchannel structure to waste via port 78A8. In this way, both the chip andlaminar channel structure are cleaned of the previous reagent materialbefore a subsequent reagent is introduced. There is also a short primingperiod prior to reagent flow to the chip, where the reagent flows towaste while the chip is again washed. This brings the multiplexer to astate of stable concentration of the new reagent, while preventingreagent from immediately entering the chip.

The parameters indicated are merely suggested values, and may beadjusted through a large range.

When the reference electrode is place upstream of the flow cell, theport 78A9 provides a convenient location for its introduction.

The need for only two-way valves is advantageous from a simplicity pointof view. Also, the valves can be located very remotely upstream of themultiplexer, and therefore can be placed in almost any location withinthe supporting instrument. The small physical size of the multiplexer,having no integrated valves or other bulky structures, suggests that itcan be located directly at the chip location, greatly reducing the totalcommon volume and providing high spatial and temporal gradients betweenwash solution and reagent.

Also feasible is a “two-dimensional” (i.e., thin, disc-like) version ofthis circuit that would allow tighter packing of reagent inputs, asindicated in FIGS. 80A and 80B at 80-1. This would be particularlyuseful if a very large number of reagents were required. Instead offlowing in only two opposite directions of a linear channel, such adevice will permit radial flow across a circular channel. A shallowrestrictive ring 80-2 replaces the restriction channels. A relativelydeep and low resistance ring on the outermost section can serve as thewaste output. Reagent inputs are supplied at 80-4 and the output istaken at 80-5.

A variation of this two-dimensional structure can be made which does notrely on laminar flow to separate out the diffuse effluent. The reagentinputs are essentially packed in a single circle centered on the chipoutput. Instead of a free and open two-dimensional channel throughoutthe circuit, narrow channels connect the reagent inputs to both the chipoutput and waste ring. When a particular reagent input flows, it entersthe central node and both exits to the chip and sweeps past the otherreagent inputs on its way to the waste ring. Diffuse effluent from thoseports enters the stream to waste, but cannot diffuse upstream toward thechip output. The relative fluidic resistances of the various channelscan be adjusted for various performances characteristics (effluentisolation, waste rate minimization, etc.).

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A method for sequencing a nucleic acid comprising: contacting andincorporating known nucleotides into a plurality of identical nucleicacids in a reaction chamber in contact with or capacitively coupled toan ISFET, wherein the nucleic acids are covalently bound to a singlebead in the reaction chamber, and detecting hydrogen ions released uponnucleotide incorporation in the presence of no or limited bufferingactivity.
 2. The method of claim 1, wherein the ISFET is in an ISFETarray that comprises 256 ISFET.
 3. The method of claim 1, wherein thereaction chamber comprises a solution having no buffer or low bufferconcentration.
 4. The method of claim 1, wherein the reaction chambercomprises a solution having a buffering inhibitor.
 5. The method ofclaim 1, wherein the single bead is at least 90% saturated with nucleicacids.
 6. The method of claim 1, wherein the nucleic acids aresequencing primers.
 7. The method of claim 6, wherein the nucleic acidsare hybridized to template nucleic acids.
 8. The method of claim 6,wherein the nucleic acids are hybridized to concatemers of identicaltemplate nucleic acids.
 9. The method of claim 1, wherein the nucleicacids are self-priming template nucleic acids. 10.-154. (canceled)